Compounds and methods for modulating adhesion molecule function

ABSTRACT

Modulating agents and methods for enhancing or inhibiting cadherin-mediated functions are provided. The modulating agents comprise at least an HAV binding motif, an analogue or peptidomimetic thereof, or an antibody or fragment thereof that specifically binds to such a motif. Modulating agents may additionally comprise one or more cell adhesion recognition sequences recognized by cadherins and/or other adhesion molecules. Such modulating agents may, but need not, be linked to a targeting agent, drug and/or support material.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/956,748 filedOct. 1, 2004; which is a continuation of U.S. Ser. No. 10/193,653 filedJul. 10, 2002, now U.S. Pat. No. 6,806,255; which is a continuation ofU.S. Ser. No. 09/351,048 filed Jul. 9, 1999, now U.S. Pat. No.6,472,368; which is a continuation-in-part of U.S. Ser. No. 09/113,977,filed Jul. 10, 1998, now U.S. Pat. No. 6,277,824.

TECHNICAL FIELD

The present invention relates generally to methods for modulatingcadherin-mediated processes, and more particularly to the use ofmodulating agents comprising a cadherin cell adhesion recognitionsequence, or an antibody that specifically recognizes such a sequence,for inhibiting or enhancing functions such as cell adhesion.

BACKGROUND OF THE INVENTION

Cell adhesion is a complex process that is important for maintainingtissue integrity and generating physical and permeability barrierswithin the body. All tissues are divided into discrete compartments,each of which is composed of a specific cell type that adheres tosimilar cell types. Such adhesion triggers the formation ofintercellular junctions (i.e., readily definable contact sites on thesurfaces of adjacent cells that are adhering to one another), also knownas tight junctions, gap junctions and belt desmosomes. The formation ofsuch junctions gives rise to physical and permeability barriers thatrestrict the free passage of cells and other biological substances fromone tissue compartment to another. For example, the blood vessels of alltissues are composed of endothelial cells. In order for components inthe blood to enter a given tissue compartment, they must first pass fromthe lumen of a blood vessel through the barrier formed by theendothelial cells of that vessel. Similarly, in order for substances toenter the body via the gut, the substances must first pass through abarrier formed by the epithelial cells of that tissue. To enter theblood via the skin, both epithelial and endothelial cell layers must becrossed.

Cell adhesion is mediated by specific cell surface adhesion molecules(CAMs). There are many different families of CAMs, including theimmunoglobulin, integrin, selectin and cadherin superfamilies, and eachcell type expresses a unique combination of these molecules. Cadherinsare a rapidly expanding family of calcium-dependent CAMs (Munro et al.,In: Cell Adhesion and Invasion in Cancer Metastasis, P. Brodt, ed., pp.17-34, RG Landes Co. (Austin Tex., 1996). The classical cadherins(abbreviated CADs) are integral membrane glycoproteins that generallypromote cell adhesion through homophilic interactions (a CAD on thesurface of one cell binds to an identical CAD on the surface of anothercell), although CADs also appear to be capable of forming heterotypiccomplexes with one another under certain circumstances and with loweraffinity. Cadherins have been shown to regulate epithelial, endothelial,neural and cancer cell adhesion, with different CADs expressed ondifferent cell types. N (neural)—cadherin is predominantly expressed byneural cells, endothelial cells and a variety of cancer cell types. E(epithelial)—cadherin is predominantly expressed by epithelial cells.Other CADs are P (placental)—cadherin, which is found in human skin andR (retinal)—cadherin. A detailed discussion of the classical cadherinsis provided in Munro S B et al., 1996, In: Cell Adhesion and Invasion inCancer Metastasis, P. Brodt, ed., pp. 17-34 (RG Landes Company, AustinTex.).

The structures of the CADs are generally similar. As illustrated in FIG.1, CADs are composed of five extracellular domains (EC1-EC5), a singlehydrophobic domain (TM) that transverses the plasma membrane (PM), andtwo cytoplasmic domains (CP1 and CP2). The calcium binding motifs DXNDN(SEQ ID NO:1), DXD and LDRE (SEQ ID NO:2) are interspersed throughoutthe extracellular domains. The first extracellular domain (EC1) containsthe classical cadherin cell adhesion recognition (CAR) sequence, HAV(His-Ala-Val), along with flanking sequences on either side of the CARsequence that may play a role in conferring specificity. Syntheticpeptides containing the CAR sequence and antibodies directed against theCAR sequence have been shown to inhibit CAD-dependent processes (Munroet al., supra; Blaschuk et al., J. Mol. Biol. 211:679-82, 1990; Blaschuket al., Develop. Biol. 139:227-29, 1990; Alexander et al., J. Cell.Physiol. 156:610-18, 1993). However, the determination of thethree-dimensional solution and crystal structures of the EC1 domain ofclassical cadherins (Overduin et al., Science 267:386-389, 1995; Shapiroet al., Nature 374:327-337, 1995) suggest that amino acid residues otherthan HAV may be directly involved in mediating the interactions betweencadherins.

Although cell adhesion is required for certain normal physiologicalfunctions, there are situations in which the level of cell adhesion isundesirable. For example, many pathologies (such as autoimmune diseases,cancer and inflammatory diseases) involve abnormal cellular adhesion.Cell adhesion may also play a role in graft rejection. In suchcircumstances, modulation of cell adhesion may be desirable.

In addition, permeability barriers arising from cell adhesion createdifficulties for the delivery of drugs to specific tissues and tumorswithin the body. For example, skin patches are a convenient tool foradministering drugs through the skin. However, the use of skin patcheshas been limited to small, hydrophobic molecules because of theepithelial and endothelial cell barriers. Similarly, endothelial cellsrender the blood capillaries largely impermeable to drugs, and theblood/brain barrier has hampered the targeting of drugs to the centralnervous system. In addition, many solid tumors develop internal barriersthat limit the delivery of anti-tumor drugs and antibodies to innercells.

Attempts to facilitate the passage of drugs across such barriersgenerally rely on specific receptors or carrier proteins that transportmolecules across barriers in vivo. However, such methods are ofteninefficient, due to low endogenous transport rates or to the poorfunctioning of a carrier protein with drugs. While improved efficiencyhas been achieved using a variety of chemical agents that disrupt celladhesion, such agents are typically associated with undesirableside-effects, may require invasive procedures for administration and mayresult in irreversible effects.

Accordingly, there is a need in the art for compounds that modulate celladhesion and improve drug delivery across permeability barriers withoutsuch disadvantages. The present invention fulfills this need and furtherprovides other related advantages.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for modulatingcadherin-mediated functions. Within certain aspects, the presentinvention provides cell adhesion modulating agents capable of binding tothe cadherin CAR sequence HAV, wherein the agent does not comprise anantibody or antigen-binding fragment thereof.

Within related aspects, the present invention provides cell adhesionmodulating agents, comprising: (a) an HAV-BM sequence or peptidomimeticthereof; (b) a polynucleotide encoding an HAV-BM sequence; or (c) anantibody or antigen-binding fragment thereof that specifically binds toan HAV-BM sequence; wherein the agent modulates a cadherin-mediatedprocess. Within certain specific embodiments, the HAV-BM sequence is:(a) Ile/Val-Phe-Aaa-Ile-Baa-Caa-Daa-Ser/Thr-Gly-Eaa-Leu/Met (SEQ IDNO:3), wherein Aaa, Baa, Caa, Daa and Eaa are independently selectedfrom the group consisting of amino acid residues; (b)Trp-Leu-Aaa-Ile-Asp/Asn-Baa-Caa-Daa-Gly-Gln-Ile (SEQ ID NO:4), whereinAaa, Baa, Caa and Daa are independently selected from the groupconsisting of amino acid residues; or (c) an analogue of any of theforegoing sequences that retains at least seven consecutive amino acidresidues, wherein the ability of the analogue to modulate acadherin-mediated process is not diminished. For example, a celladhesion modulating agent may comprise an HAV-BM sequence is selectedfrom the group consisting of: IFIINPISGQL (SEQ ID NO:5), IFILNPISGQL(SEQ ID NO:6), VFAVEKETGWL (SEQ ID NO:7), VFSINSMSGRM (SEQ ID NO:8),VFIIERETGWL (SEQ ID NO:9), VFTIEKESGWL (SEQ ID NO:10), VFNIDSMSGRM (SEQID NO:11), WLKIDSVNGQI (SEQ ID NO:12), WLKIDPVNGQI (SEQ ID NO:13),WLAMDPDSGQV (SEQ ID NO:14), WLHINATNGQI (SEQ ID NO:15), WLEINPDTGAI (SEQID NO:16), WLAVDPDSGQI (SEQ ID NO:17), WLEINPETGAI (SEQ ID NO:18),WLHINTSNGQI (SEQ ID NO:19), NLKIDPVNGQI (SEQ ID NO:20), LKIDPVNGQI (SEQID NO:21) and analogues of the foregoing sequences that retain at leastseven consecutive residues (e.g., INPISGQ (SEQ ID NO:22), LNPISGQ (SEQID NO:23), IDPVSGQ (SEQ ID NO:24) or KIDPVNGQ (SEQ ID NO:25)), whereinthe ability of the analogue to modulate a cadherin-mediated process isnot diminished. Alternatively, a modulating agent may comprise an HAV-BMsequence that comprises at least five consecutive residues of a peptideselected from the group consisting of INPISGQ (SEQ ID NO:22), LNPISGQ(SEQ ID NO:23), NLKIDPVNGQI (SEQ ID NO:20) and WLKIDPVNGQI (SEQ IDNO:13). For example, the agent may comprise a sequence selected from thegroup consisting of PISGQ (SEQ ID NO:26), PVNGQ (SEQ ID NO:27), PVSGR(SEQ ID NO:28), IDPVN (SEQ ID NO:29), INPIS (SEQ ID NO:30) and KIDPV(SEQ ID NO:31). Within such modulating agents, an HAV-BM sequence may bepresent within a linear peptide or a cyclic peptide. Certain modulatingagents comprise a cyclic peptide having one of the formulas:

wherein X₁, and X₂ are optional, and if present, are independentlyselected from the group consisting of amino acid residues andcombinations thereof in which the residues are linked by peptide bonds,and wherein X₁ and X₂ independently range in size from 0 to 10 residues,such that the sum of residues contained within X₁ and X₂ ranges from 1to 12; wherein Y₁ and Y₂ are independently selected from the groupconsisting of amino acid residues, and wherein a covalent bond is formedbetween residues Y₁ and Y₂; and wherein Z₁ and Z₂ are optional, and ifpresent, are independently selected from the group consisting of aminoacid residues and combinations thereof in which the residues are linkedby peptide bonds. Such cyclic peptides may contain modifications. Forexample, Y₁ may comprise an N-acetyl group and/or Y₂ may comprise aC-terminal amide group. Cyclization may be achieved in any of a varietyof ways, such as covalent linkage of Y₁ and Y₂ via a disulfide, amide orthioether bond.

Within certain embodiments, modulating agents as described above may belinked to one or more of a drug, a solid support, a detectable marker ora targeting agent.

Within other embodiments, a modulating agents as described above mayfurther comprise one or more of: (a) a cell adhesion recognitionsequence other than an HAV-BM sequence, wherein the cell adhesionrecognition sequence is separated from any HAV-BM sequence(s) by alinker; and/or (b) an antibody or antigen-binding fragment thereof thatspecifically binds to a cell adhesion recognition sequence other than anHAV-BM sequence. For example, the adhesion molecule may be selected fromthe group consisting of cadherins, integrins, occludin, N-CAM,desmogleins, desmocollins, fibronectin, laminin and other extracellularmatrix proteins.

Within further aspects, the present invention provides pharmaceuticalcompositions comprising a cell adhesion modulating agent as describedabove, in combination with a pharmaceutically acceptable carrier. Suchcompositions may further comprise one or more of a drug and/or amodulator of cell adhesion, wherein the modulator comprises one or moreof: (a) a peptide comprising a cell adhesion recognition sequence otherthan an HAV-BM sequence; and/or (b) an antibody or antigen-bindingfragment thereof that specifically binds to a cell adhesion recognitionsequence other than an HAV-BM sequence. For example, the adhesionmolecule may be selected from the group consisting of cadherins,integrins, occludin, N-CAM, desmogleins, desmocollins, fibronectin,laminin and other extracellular matrix proteins.

The present invention further provides, within other aspects, methodsfor modulating a cadherin-mediated function, comprising contacting acadherin-expressing cell with a cell adhesion modulating agent asdescribed above. Cadherin-mediated functions include cell adhesion,neurite outgrowth, Schwann cell migration and synaptic stability.Cadherin-expressing cells include epithelial cells, endothelial cells,neural cells, tumor cells and lymphocytes. Within such aspects, the celladhesion modulating agent may inhibit or enhance a cadherin-mediatedfunction.

Within other aspects, the present invention provides methods forreducing unwanted cellular adhesion in a mammal, comprisingadministering to a mammal a modulating agent as described above, whereinthe modulating agent inhibits cadherin-mediated cell adhesion. The cellmay be selected from the group consisting of epithelial cells,endothelial cells, neural cells, tumor cells and lymphocytes.

The present invention further provides, within other aspects, methodsfor enhancing the delivery of a drug through the skin of a mammal,comprising contacting epithelial cells of a mammal with a drug and amodulating agent as described above, wherein the step of contacting isperformed under conditions and for a time sufficient to allow passage ofthe drug across the epithelial cells, and wherein the modulating agentinhibits cadherin-mediated cell adhesion. The modulating agent may, butneed not, be linked to the drug and may, within certain embodiments,pass into the blood stream of the mammal. The step of contacting may beperformed via a skin patch comprising the modulating agent and the drug.

Within further aspects, methods are provided for enhancing the deliveryof a drug to a tumor in a mammal, comprising administering to a mammal amodulating agent as described above, wherein the modulating agentinhibits cadherin-mediated cell adhesion. Suitable tumors include, butare not limited to, bladder tumors, ovarian tumors and melanomas, andthe modulating agent may be administered to the tumor or systemically.

Within other aspects, the present invention provides methods fortreating cancer and/or inhibiting metastasis in a mammal, comprisingadministering to a mammal a modulating agent as described above, whereinthe modulating agent inhibits cadherin-mediated cell adhesion. Themammal may be afflicted with a cancer such as a carcinoma, leukemia ormelanoma, and the modulating agent may be administered to the tumor orsystemically.

The present invention further provides, within other aspects, methodsfor inducing apoptosis in a cadherin-expressing cell, comprisingcontacting a cadherin-expressing cell with a modulating agent asdescribed above, wherein the modulating agent inhibits cadherin-mediatedcell adhesion.

Within other aspects, methods are provided for inhibiting angiogenesisin a mammal, comprising administering to a mammal a modulating agent asdescribed above, wherein the modulating agent inhibits cadherin-mediatedcell adhesion.

The present invention further provides, within other aspects, methodsfor enhancing drug delivery to the central nervous system of a mammal,comprising administering to a mammal a modulating agent as describedabove, wherein the modulating agent inhibits cadherin-mediated celladhesion.

Within further aspects, the present invention provides methods forfacilitating wound healing in a mammal, comprising contacting a wound ina mammal with a modulating agent as described above, wherein themodulating agent enhances cadherin-mediated cell adhesion.

Methods are also provided, within other aspects, for enhancing adhesionof foreign tissue implanted within a mammal, comprising contacting asite of implantation of foreign tissue in a mammal with a modulatingagent as described above, wherein the modulating agent enhancescadherin-mediated cell adhesion. Such foreign tissue may be a skin graftor organ implant. Within certain embodiments, the modulating agent islinked to a support material.

The present invention further provides, in other aspects, methods forenhancing and/or directing neurite outgrowth, comprising contacting aneuron with a modulating agent as described above, wherein themodulating agent enhances cadherin-mediated cell adhesion.

Within other aspects, the present invention provides methods fortreating spinal cord injuries in a mammal, comprising administering to amammal a modulating agent as described above, wherein the modulatingagent enhances cadherin-mediated cell adhesion.

Methods are also provided, within further aspects, for treating ademyelinating neurological disease such as multiple sclerosis in amammal, comprising administering to a mammal a modulating agent asdescribed above. Within certain embodiments, the modulating agent isadministered by implantation with Schwann cells, oligodendrocyteprogenitor cells and/or oligodendrocytes.

Within further aspects, methods are provided for modulating the immunesystem of a mammal, comprising administering to a mammal a modulatingagent as described above, wherein the modulating agent inhibitscadherin-mediated cell adhesion.

Within other aspects, the present invention provides methods forpreventing pregnancy in a mammal, comprising administering to a mammal amodulating agent as described above, wherein the modulating agentinhibits cadherin-mediated cell adhesion.

Methods are further provided for increasing vasopermeability in amammal, comprising administering to a mammal a modulating agent asdescribed above, wherein the modulating agent inhibits cadherin-mediatedcell adhesion.

Within further aspects, the present invention provides methods forinhibiting synaptic stability in a mammal, comprising administering to amammal a modulating agent as described above, wherein the modulatingagent inhibits cadherin-mediated cell adhesion.

The present invention further provides methods for detecting thepresence of cadherin-expressing cells in a sample, comprising: (a)contacting a sample with an antibody or antigen-binding fragment thereofthat binds to an HAV-BM sequence under conditions and for a timesufficient to allow formation of an antibody-cadherin complex; and (b)detecting the level of antibody-cadherin complex, and therefromdetecting the presence of cadherin expressing cells in a sample. Theantibody may be linked to a support material or a detectable marker suchas a fluorescent marker. In certain embodiments, the step of detectingis performed using fluorescence activated cell sorting.

The present invention also provides, within further aspects, kits forenhancing transdermal drug delivery, comprising: (a) a skin patch; and(b) a modulating agent as described above. The skin patch may beimpregnated with the modulating agent, and the kit may further comprisea drug.

Kits for detecting the presence of cadherin-expressing cells in a sampleare also provided. Such kits may comprise: (a) an antibody orantigen-binding fragment thereof that specifically binds to an HAV-BMsequence; and (b) a detection reagent.

Within other aspects, the present invention provides methods foridentifying a compound capable of modulating cadherin-mediated celladhesion, comprising: (a) contacting an antibody or antigen-bindingfragment thereof that specifically binds to an HAV-BM sequence with atest compound; and (b) detecting the level of antibody or fragment thatbinds to the test compound, and therefrom identifying a compound capableof modulating cadherin-mediated cell adhesion.

Methods are also provided, within other aspects, for facilitating bloodsampling in a mammal, comprising contacting epithelial cells of a mammalwith a cell adhesion modulating agent as described above, wherein themodulating agent inhibits cadherin-mediated cell adhesion, and whereinthe step of contacting is performed under conditions and for a timesufficient to allow passage of one or more blood components across theepithelial cells. The step of contacting may be performed via a skinpatch comprising the modulating agent, and the skin patch may furthercomprise a reagent for detecting a blood component of interest. Withincertain embodiments, the epithelial cells are skin cells or gum cells.

Within related aspects, the present invention provides kits for samplingblood via the skin or gum of a mammal, comprising: (a) a skin patch; (b)a cell adhesion modulating agent comprising a cyclic peptide thatcomprises a cadherin CAR sequence; and (c) a reagent for detecting ablood component of interest. The skin patch may be impregnated with thecell adhesion modulating agent.

Within other aspects, the present invention provides methods forscreening for a compound that interacts with an HAV-BM sequence,comprising the steps of: (a) contacting a candidate compound with anHAV-BM sequence; and (b) evaluating the ability of the candidatecompound to bind to the HAV-BM sequence, and therefrom determiningwhether the candidate compound interacts with an HAV-BM sequence. Withincertain embodiments, the ability of the candidate compound to bind tothe HAV-BM sequence is evaluated using an affinity column or a Westernblot analysis. Within certain embodiments, the candidate compound isencoded by a polynucleotide in an expression library, or the candidatecompound is a cellular protein, and step (b) is performed using wholecells.

These and other aspects of the invention will become evident uponreference to the following detailed description and attached drawings.All references disclosed herein are hereby incorporated by reference intheir entirety as if each were individually noted for incorporation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram depicting the structure of classical CADs. The fiveextracellular domains are designated EC1-EC5, the hydrophobic domainthat transverses the plasma membrane (PM) is represented by TM, and thetwo cytoplasmic domains are represented by CP1 and CP2. The calciumbinding motifs are shown by DXNDN (SEQ ID NO:1), DXD and LDRE (SEQ IDNO:2). The CAR sequence, HAV, is shown within EC1. The sequences,INPISGQ (SEQ ID NO:22) and LKIDPVNGQI (SEQ ID NO:21) are shown withinEC1 and EC4, respectively. Cytoplasmic proteins β-catenin (β), α-catenin(α) and α-actinin (ACT), which mediate the interaction between CADs andmicrofilaments (MF) are also shown.

FIG. 2 provides the amino acid sequences of mammalian classical cadherinEC1 domains: human N-cadherin (SEQ ID NO:32), mouse N-cadherin (SEQ IDNO:33), cow N-cadherin (SEQ ID NO:34), human E-cadherin (SEQ ID NO:35),mouse E-cadherin (SEQ ID NO:36), human P-cadherin (SEQ ID NO:37), mouseP-cadherin (SEQ ID NO:38), human R-cadherin (SEQ ID NO:39) and mouseR-cadherin (SEQ ID NO:40).

FIG. 3 provides the amino acid sequences of mammalian classical cadherinEC4 domains: human N-cadherin (SEQ ID NO:41), mouse N-cadherin (SEQ IDNO:42), cow N-cadherin (SEQ ID NO:43), human E-cadherin (SEQ ID NO:44),mouse E-cadherin (SEQ ID NO:45), human P-cadherin (SEQ ID NO:46), mouseP-cadherin (SEQ ID NO:47), human R-cadherin (SEQ ID NO:48) and mouseR-cadherin (SEQ ID NO:49).

FIGS. 4A-4E provide structures of representative cyclic peptidemodulating agents (SEQ ID NOs: 51-63 and 85).

FIG. 5 is a graph showing the mean neurite length measured for neuronscultured on monolayers of 3T3 cells or 3T3 cells expressing N-cadherinin media containing varying concentrations of the linear peptideH-WLKIDPVNGQI-OH (SEQ ID NO:13; designated N-CAD-CHD2).

FIG. 6 is a graph showing the mean neurite length measured for neuronscultured on monolayers of either 3T3 cells, 3T3 cells expressingN-cadherin, 3T3 cells expressing NCAM, or 3T3 cells expressing L1 inmedia containing the linear peptide H-WLKIDPVNGQI-OH (SEQ ID NO:13;designated N-CAD-CHD2) at a concentration of 250 μg/ml.

FIG. 7 is a graph illustrating the binding of the peptideH-WLKIDPVNGQI-OH (SEQ ID NO:13) at various concentrations to a flow cellcoated with an N-cadherin-Fc chimera or human IgG1. The peptideH-WLKIDPVNGQI-OH (SEQ ID NO:13) was passed over both flow cells at aconcentration of either 250, 500 or 1000 μg/ml. The results show theassociation of the peptide to the flow cell coated with the N-cadherinFc chimera, with the binding to the control flow cell (coated with humanIgG1) automatically subtracted.

FIGS. 8A and 8B are photographs showing monolayer cultures of humanovarian cancer cells (SKOV3) in the presence (FIG. 8B) and absence (FIG.8A) of the peptide N-Ac-INPISGQ-NH₂ (SEQ ID NO:22). FIG. 8B shows thecells 24 hours after being cultured in the presence of 1 mg/mL ofN-Ac-INPISGQ (SEQ ID NO:22).

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides methods for modulatingcadherin-mediated processes, such as cell adhesion. The presentinvention is based upon the identification of a previously unknown celladhesion recognition (CAR) sequence in classical cadherins. This CARsequence is referred to herein as the “HAV binding motif” (or “HAV-BM”).The HAV-BM appears to interact directly with the HAV CAR sequence and/orflanking regions in homophilic and heterophilic interactions.

In general, to modulate cadherin-mediated cell adhesion, a cell thatexpresses a classical cadherin is contacted with a cell adhesionmodulating agent (also referred to herein as a “modulating agent”)either in vivo or in vitro. A modulating agent may comprise one or moreHAV-BM sequences (which may be native sequences or analogues thereof) ora peptidomimetic of such a sequence, with or without one or moreadditional CAR sequences (which may be derived from classical cadherinsor from other adhesion molecules), as described below. HAV-BM sequencesmay be present within a linear or cyclic peptide. Alternatively, or inaddition, a modulating agent may comprise a polynucleotide encoding apeptide comprising one or more HAV-BM sequences (such that the encodedpeptide is produced in vivo) and/or a substance (such as an antibody orantigen-binding fragment thereof) that specifically binds an HAV-BMsequence.

Certain methods provided herein employ cell adhesion modulating agentsfor inhibiting or enhancing cadherin-mediated cell adhesion. Inhibitionof cell adhesion may generally be used, for example, to treat diseasesor other conditions characterized by undesirable cell adhesion or tofacilitate drug delivery to a specific tissue or tumor. Within otheraspects, the methods provided herein may be used to enhance celladhesion (e.g., to supplement or replace stitches or to facilitate woundhealing). Within still further aspects, methods are provided forenhancing and/or directing neurite outgrowth.

Cell Adhesion Modulating Agents

As noted above, the term “cell adhesion modulating agent,” as usedherein, generally refers to a compound that is capable of binding to aclassical cadherin (i.e., the compound interacts detectably with one ormore amino acid residues within a classical cadherin such that acadherin-mediated process is modulated, as described herein).Preferably, a modulating agent binds in or near a classical cadherin CARsequence HAV (i.e., the agent interacts detectably with one or moreamino acid residues present within the HAV sequence and/or one or moreamino acid residues present within ten amino acid residues, and morepreferably within five amino acid residues, of the HAV sequence in anative cadherin). Within specific embodiments, a modulating agentcomprises at least one of the following:

(a) an HAV-BM sequence (i.e., a native HAV-BM sequence or an analoguethereof), or a peptidomimetic thereof;

(b) a polynucleotide encoding an HAV-BM sequence; or

(c) an antibody or antigen-binding fragment thereof that specificallybinds to an HAV-BM sequence.

A modulating agent may consist entirely of an HAV-BM sequence (within alinear or cyclic peptide), peptidomimetic, polynucleotide or antibody,or may additionally comprise further peptide and/or non-peptide regions.

An “HAV-BM sequence” is an HAV-binding sequence that exists in anaturally occurring cadherin, or an analogue of such a sequence in whichthe ability to modulate a cadherin-mediated process is not diminished.Such sequences generally comprise at least five amino acid residues,preferably 6-16 amino acid residues, and may be identified based onsequence homology to known HAV-BM sequences, which are provided herein,and based on the ability of a peptide comprising such a sequence to bindto an HAV sequence and modulate a cadherin-mediated function, within arepresentative assay as described herein. Within certain embodiments,the HAV-BM sequence is:

(a) Ile/Val-Phe-Aaa-Ile-Baa-Caa-Daa-Ser/Thr-Gly-Eaa-Leu/Met (SEQ IDNO:3), wherein Aaa, Baa, Caa, Daa and Eaa are independently selectedfrom the group consisting of amino acid residues;

(b) Trp-Leu-Aaa-Ile-Asp/Asn-Baa-Caa-Daa-Gly-Gln-Ile (SEQ ID NO:4),wherein Aaa, Baa, Caa and Daa are independently selected from the groupconsisting of amino acid residues; or

(c) an analogue of any of the foregoing sequences that retains at leastseven consecutive amino acid residues.

Representative known HAV-BM sequences are provided in Table 1. Thesesequences are not intended to limit the scope of HAV-BM sequencesencompassed by the present invention. In particular, a modulating agentmay comprise a portion or other analogue of such sequences, providedthat the ability of the analogue to modulate a cadherin-mediatedfunction is not substantially diminished. TABLE I Representative HAV-BMSequences Cadherin HAV-BM EC1 Domains BTCADHN IFIINPISGQL (SEQ ID NO:5)HSNCADHER IFILNPISGQL (SEQ ID NO:6) HSPCAD VFAVEKETGWL (SEQ ID NO:7)HUMCA4A VFSINSMSGRM (SEQ ID NO:8) HUMUVOECAD VFIIERETGWL (SEQ ID NO:9)MMCADHP VFTIEKESGWL (SEQ ID NO:10) MMECADH VFIIERETGWL (SEQ ID NO:9)MMRCADA VFNIDSMSGRM (SEQ ID NO:11) MUSCADNA IFIINPISGQL (SEQ ID NO:5)CONSENSUS IFXIXXXSGXL (SEQ ID NO:3) V      T  M EC4 Domains BTCADHNWLKIDSVNGQI (SEQ ID NO:12) HSNCADHER WLKIDPVNGQI (SEQ ID NO:13) HSPCADWLAMDPDSGQV (SEQ ID NO:14) HUMCA4A WLHINATNGQI (SEQ ID NO:15) HUMUVOECADWLEINPDTGAI (SEQ ID NO:16) MMCADHP WLAVDPDSGQI (SEQ ID NO:17) MMECADHWLEINPETGAI (SEQ ID NO:18) MMRCADA WLHINTSNGQI (SEQ ID NO:19) MUSCADNAWLKIDPVNGQI (SEQ ID NO:13) CONSENSUS WLXIDXXXGQI (SEQ ID NO:4)     N

Within certain specific embodiments, the HAV-BM sequence comprisesINPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ (SEQ ID NO:24)or KIDPVNGQ (SEQ ID NO:25). For example, HAV-BM sequences include, butare not limited to N-Ac-NLKIDPVNGQI-NH₂ (SEQ ID NO:20) andH-LKIDPVNGQI-OH (SEQ ID NO:21).

Within other embodiments, an HAV-BM sequence may comprise at least fiveconsecutive residues of one of the following peptides: INPISGQ (SEQ IDNO:22), LNPISGQ (SEQ ID NO:23), NLKIDPVNGQI (SEQ ID NO:20) andWLKIDPVNGQI (SEQ ID NO:13). For example, a modulating agent may comprisethe sequence PISGQ (SEQ ID NO:26), PVNGQ (SEQ ID NO:27), PVSGR (SEQ IDNO:28), KIDPV (SEQ ID NO:31), IDPVN (SEQ ID NO:29), INPIS (SEQ ID NO:30)or KIDPVN (SEQ ID NO:50). As noted above, within any of the aboveembodiments, an HAV-BM sequence may be present within a cyclic peptide,such as PVNGQ (SEQ ID NO:51), PISGQ (SEQ ID NO:52), PVSGR (SEQ IDNO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ ID NO:55), IDPVN (SEQ IDNO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ ID NO:58), CPISGQC (SEQ IDNO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ ID NO:61), CIDPVNC (SEQ IDNO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ ID NO:85), CINPC (SEQ IDNO:86) or CINPIC (SEQ ID NO:87), in which cyclization is indicated bythe underline.

Other HAV-BM sequences include sequences in which a native sequence ismodified. For example, the peptides H-LKIDPANGQI-OH (SEQ ID NO:64) andH-LKIDAVNGQI-OH (SEQ ID NO:65) comprise HAV-BM sequences.

As noted above, the present invention further contemplates native HAV-BMsequences from other cadherins not specifically recited herein.Additional native HAV-BM sequences may be identified based upon sequencesimilarity to one or more of the native HAV-BMs provided herein. Ingeneral, a native HAV-BM sequence should retain at least three aminoacid residues of a native HAV-BM provided herein, and a total of atleast seven amino acid residues should be identical or containconservative substitutions. A “conservative substitution” is one inwhich an amino acid is substituted for another amino acid that hassimilar properties, such that one skilled in the art of peptidechemistry would expect the secondary structure and hydropathic nature ofthe polypeptide to be substantially unchanged. Amino acid substitutionsmay generally be made on the basis of similarity on polarity, charge,solubility, hydrophobicity, hydrophilicity and/or the amphipathic natureof the residues. For example, negatively charged amino acids includeaspartic acid and glutamic acid; positively charged amino acids includelysine and arginine; and amino acids with uncharged polar head groupshaving similar hydrophilicity values include leucine, isoleucine andvaline; glycine and alanine; asparagine and glutamine; and serine,threonine, phenylalanine and tyrosine. Other groups of amino acids thatmay represent conservative changes include: (1) ala, pro, gly, glu, asp,gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, Ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. The criticaldetermining features of a native HAV-BM are the ability to bind to anHAV sequence and the ability to modulate a cadherin-mediated function.Such abilities may be evaluated using the representative assays providedherein.

As noted above, modulating agents as described herein may comprise anative HAV-BM sequence, or an analogue or peptidomimetic thereof. Ananalogue generally retains at least three amino acid residues of anative HAV-BM, and binds to an HAV sequence and modulates acadherin-mediated function as described below. In particular, ananalogue should bind to a classical cadherin and modulate acadherin-mediated function at least as well as a native HAV-BM sequencewithin at least one of the assays provided herein. A peptidomimetic is anon-peptide compound that is structurally similar to an HAV-BM sequence,such that it binds to HAV sequences and modulates a cadherin-mediatedfunction as described below. Such peptidomimetics may be designed basedon techniques that evaluate the three dimensional structure of apeptide. For example, nuclear magnetic resonance (NMR) and computationaltechniques may be used to determine the conformation of an HAV-BMsequence. NMR is widely used for structural analyses of both peptidyland non-peptidyl compounds. Nuclear Overhauser Enhancements (NOE's),coupling constants and chemical shifts depend on the conformation of acompound. NOE data provides the interproton distance between protonsthrough space and can be used to calculate of the lowest energyconformation for the HAV-BM sequence. This information can then be usedto design peptidomimetics of the preferred conformation. Linear peptidesin solution exist in many conformations. By using conformationalrestriction techniques it is possible to fix the peptide in the activeconformation. Conformational restriction can be achieved by i)introduction of an alkyl group such as a methyl which stericallyrestricts free bond rotation; ii) introduction of unsaturation whichfixes the relative positions of the terminal and geminal substitutents;and/or iii) cyclization, which fixes the relative positions of thesidechains. Peptidomimetics of an HAV-BM sequence may be synthesizedwhere one or more of the amide linkages has been replaced by isosteres,substitutents or groups which have the same size or volume such as, butnot limited to, —CH₂NH—, —CSNH—, —CH₂S—, —CH═CH—, —CH₂CH₂—, —CONMe- andothers. These backbone amide linkages can be also be part of a ringstructure (i.e., lactam). Peptidomimetics of an HAV-BM sequence may bedesigned where one or more of the side chain functionalities of theHAV-BM sequence can be replaced by groups that do not necessarily havethe same size or volume, but have similar chemical and/or physicalproperties which produce similar biological responses. It should beunderstood that, within embodiments described below, an analogue orpeptidomimetic may be substituted for an HAV-BM sequence.

Modulating agents, or peptide portions thereof, may generally comprisefrom 5 to about 1000 amino acid residues, preferably from 6 to 50residues. When non-peptide linkers are employed, each CAR sequence ofthe modulating agent is present within a peptide that generally rangesin size from 5 to 50 residues in length, preferably from 5 to 25residues, more preferably from 5 to 16 residues and still morepreferably from 5 or 6 to 10 residues.

Modulating agents, or peptide portions thereof, may be linear or cyclicpeptides. The term “cyclic peptide,” as used herein, refers to a peptideor salt thereof that comprises (1) an intramolecular covalent bondbetween two non-adjacent residues and (2) at least one HAV-BM sequenceor an analogue thereof present within the peptide ring. Theintramolecular bond may be a backbone to backbone, side-chain tobackbone or side-chain to side-chain bond (i.e., terminal functionalgroups of a linear peptide and/or side chain functional groups of aterminal or interior residue may be linked to achieve cyclization).Preferred intramolecular bonds include, but are not limited to,disulfide, amide and thioether bonds. As noted above, in addition to oneor more HAV-BM sequence or analogue thereof, a modulating agent maycomprise additional CAR sequences, which may or may not be cadherin CARsequences, and/or antibodies or fragments thereof that specificallyrecognize a CAR sequence. Antibodies and antigen-binding fragmentsthereof are typically present in a non-cyclic portion of a modulatingagent.

The size of a cyclic peptide ring generally ranges from 4 to about 15residues, preferably from 5 to 10 residues. Additional residue(s) may bepresent on the N-terminal and/or C-terminal side of an HAV-BM sequence,and may be derived from sequences that flank a native HAV-BM sequence,with or without amino acid substitutions and/or other modifications.Additional residue(s) that may be present on the N-terminal and/orC-terminal side of an HAV-BM sequence may be derived from sequences thatflank the HAV-BM sequence within one or more naturally occurringcadherins, with or without amino acid substitutions and/or othermodifications. Flanking sequences for endogenous N-, E-, P- andR-cadherin HAV-BMs are shown in FIGS. 2 and 3, and SEQ ID NOs: 32 to 49.Alternatively, additional residues present on one or both sides of theCAR sequence(s) may be unrelated to an endogenous sequence (e.g.,residues that facilitate cyclization, purification or other manipulationand/or residues having a targeting or other function).

In certain preferred embodiments, a modulating agent comprises a cyclicpeptide having one of the following structures:

In these structures, X₁, and X₂ are optional, and if present, areindependently selected amino acid residues and combinations thereof inwhich the residues are linked by peptide bonds. In general, X₁ and X₂independently range in size from 0 to 10 residues, such that the sum ofresidues contained within X₁ and X₂ ranges from 1 to 12. Y₁ and Y₂ areindependently selected amino acid residues, and a covalent bond isformed between residues Y₁ and Y₂. Z₁ and Z₂ are optional, and ifpresent, are independently selected amino acid residues and combinationsthereof in which the residues are linked by peptide bonds.Representative examples of such structures are provided in FIGS. 4A-4D.

A modulating agent that contains sequences that flank the HAV-BMsequence on one or both sides may be specific for cell adhesion mediatedby one or more specific cadherins, resulting in tissue and/or cell-typespecificity. Suitable flanking sequences for conferring specificityinclude, but are not limited to, endogenous sequences present in one ormore naturally occurring cadherins. Modulating agents having a desiredspecificity may be identified using the representative screens providedherein.

As noted above, multiple CAR sequences may be present within amodulating agent. The total number of CAR sequences present within amodulating agent may range from 1 to a large number, such as 100,preferably from 1 to 10, and more preferably from 1 to 5. CAR sequencesthat may be included within a modulating agent are any sequencesspecifically bound by an adhesion molecule (i.e., a molecule thatmediates cell adhesion via a receptor on the cell's surface). Adhesionmolecules include members of the cadherin gene superfamily that are notclassical cadherins (e.g., proteins that do not contain an HAV sequenceand/or one or more of the other characteristics recited above forclassical cadherins), such as desmogleins (Dsg) and desmocollins (Dsc);integrins; members of the immunoglobulin supergene family, such asN-CAM; and other uncategorized transmembrane proteins, such as occludin,as well as extracellular matrix proteins such as laminin, fibronectin,collagens, vitronectin, entactin and tenascin. Within certainembodiments, preferred CAR sequences for inclusion within a modulatingagent include Arg-Gly-Asp (RGD), which is bound by integrins (seeCardarelli et al., J. Biol. Chem. 267:23159-64, 1992);Tyr-Ile-Gly-Ser-Arg (YIGSR; SEQ ID NO:66), which is bound by α6β1integrin; KYSFNYDGSE (SEQ ID NO:67), which is bound by N-CAM; the N-CAMheparin sulfate-binding site IWKHKGRDVILKKDVRF (SEQ ID NO:68), theoccludin CAR sequence LYHY (SEQ ID NO:70); a junctional adhesionmolecule CAR sequence DPK and/or one or more nonclassical cadherin CARsequences, such as the VE-cadherin CAR sequence DAE, the Dsc CARsequences IEK, VER and IER, the Dsg CAR sequences INQ, INR and LNK; andthe claudin CAR sequence IYSY (SEQ ID NO:95).

Within certain embodiments, another preferred CAR sequence is theOB-cadherin CAR sequence DDK. A variety of peptides comprising thissequence may be included, such as IDDK (SEQ ID NO:71), DDKS (SEQ IDNO:72), VIDDK (SEQ ID NO:73), IDDKS (SEQ ID NO:74), VIDDKS (SEQ IDNO:75), DDKSG (SEQ ID NO:76), IDDKSG (SEQ ID NO:77), VIDDKSG (SEQ IDNO:78), FVIDDK (SEQ ID NO:79), FVIDDKS (SEQ ID NO:80), FVIDDKSG (SEQ IDNO:81), IFVIDDK (SEQ ID NO:82), IFVIDDKS (SEQ ID NO:83), or IFVIDDKSG(SEQ ID NO:84). In certain preferred embodiments, at least one terminalamino acid residue of such a peptide is modified (e.g., the N-terminalamino group is modified by, for example, acetylation oralkoxybenzylation and/or an amide or ester is formed at the C-terminus).Certain preferred modulating agents contain modifications at the N- andC-terminal residues, such as N-Ac-IFVIDDKSG-NH₂ (SEQ ID NO:84).Analogues of any of the foregoing sequences may also be used. Ananalogue generally retains at least 50% of a native OB-cadherin CARsequence, and modulates OB-cadherin-mediated cell adhesion.

Linkers may, but need not, be used to separate CAR sequences and/orantibody sequences within a modulating agent. Linkers may also, oralternatively, be used to attach one or more modulating agents to asupport molecule or material, as described below. A linker may be anymolecule (including peptide and/or non-peptide sequences as well assingle amino acids or other molecules), that does not contain a CARsequence and that can be covalently linked to at least two peptidesequences. Using a linker, HAV-BM-containing peptides and other peptideor protein sequences may be joined head-to-tail (i.e., the linker may becovalently attached to the carboxyl or amino group of each peptidesequence), head-to-side chain and/or tail-to-side chain. Modulatingagents comprising one or more linkers may form linear or branchedstructures. Within one embodiment, modulating agents having a branchedstructure comprise three different CAR sequences, such as RGD, YIGSR(SEQ ID NO:66) and an HAV-BM sequence. Within another embodiment,modulating agents having a branched structure may comprise RGD, YIGSR(SEQ ID NO:66), an HAV-BM sequence and KYSFNYDGSE (SEQ ID NO:67). In athird embodiment, modulating agents having a branched structure comprisean HAV-BM sequence, one or more Dsc CAR sequences, one or more Dsg CARsequence and LYHY (SEQ ID NO:70).

Linkers preferably produce a distance between CAR sequences between 0.1to 10,000 nm, more preferably about 0.1-400 nm. A separation distancebetween recognition sites may generally be determined according to thedesired function of the modulating agent. For inhibitors of celladhesion, the linker distance between HAV-BM sequences should be small(0.1-400 nm). For enhancers of cell adhesion, the linker distancebetween HAV-BM sequences should be 400-10,000 nm. One linker that can beused for such purposes is (H₂N(CH₂)_(n)CO₂H)_(m), or derivativesthereof, where n ranges from 1 to 10 and m ranges from 1 to 4000. Forexample, if glycine (H₂NCH₂CO₂H) or a multimer thereof is used as alinker, each glycine unit corresponds to a linking distance of 2.45angstroms, or 0.245 nm, as determined by calculation of its lowestenergy conformation when linked to other amino acids using molecularmodeling techniques. Similarly, aminopropanoic acid corresponds to alinking distance of 3.73 angstroms, aminobutanoic acid to 4.96angstroms, aminopentanoic acid to 6.30 angstroms and amino hexanoic acidto 6.12 angstroms. Other linkers that may be used will be apparent tothose of ordinary skill in the art and include, for example, linkersbased on repeat units of 2,3-diaminopropanoic acid, lysine and/orornithine. 2,3-Diaminopropanoic acid can provide a linking distance ofeither 2.51 or 3.11 angstroms depending on whether the side-chain aminoor terminal amino is used in the linkage. Similarly, lysine can providelinking distances of either 2.44 or 6.95 angstroms and ornithine 2.44 or5.61 angstroms. Peptide and non-peptide linkers may generally beincorporated into a modulating agent using any appropriate method knownin the art.

Modulating agents that inhibit cell adhesion typically contain oneHAV-BM sequence or multiple HAV-BM sequences, which may be adjacent toone another (i.e., without intervening sequences) or in close proximity(i.e., separated by peptide and/or non-peptide linkers to give adistance between the CAR sequences that ranges from about 0.1 to 400nm). Within one such embodiment, a modulating agent contains two HAV-BMsequences. Such a modulating agent may additionally comprise a CARsequence for one or more different adhesion molecules (including, butnot limited to, other CAMs) and/or one or more antibodies or fragmentsthereof that bind to such sequences. Linkers may, but need not, be usedto separate such CAR sequence(s) and/or antibody sequence(s) from theHAV-BM sequence(s) and/or each other. Such modulating agents maygenerally be used within methods in which it is desirable tosimultaneously disrupt cell adhesion mediated by multiple adhesionmolecules. Within certain preferred embodiments, the second CAR sequenceis derived from fibronectin and is recognized by an integrin (i.e., RGD;see Cardarelli et al., J. Biol. Chem. 267:23159-23164, 1992), or is anoccludin CAR sequence (e.g., LYHY; SEQ ID NO:70). One or moreantibodies, or fragments thereof, may similarly be used within suchembodiments.

Modulating agents that enhance cell adhesion may contain multiple HAV-BMsequences, and/or antibodies that specifically bind to such sequences,joined by linkers as described above. Enhancement of cell adhesion mayalso be achieved by attachment of multiple modulating agents to asupport molecule or material, as discussed further below. Suchmodulating agents may additionally comprise one or more CAR sequence forone or more different adhesion molecules (including, but not limited to,other CAMs) and/or one or more antibodies or fragments thereof that bindto such sequences, to enhance cell adhesion mediated by multipleadhesion molecules.

As noted above, modulating agents may be polypeptides or salts thereof,containing only amino acid residues linked by peptide bonds, or maycontain non-peptide regions, such as linkers. Peptide regions of amodulating agent may comprise residues of L-amino acids, D-amino acids,or any combination thereof. Amino acids may be from natural ornon-natural sources, provided that at least one amino group and at leastone carboxyl group are present in the molecule; α- and β-amino acids aregenerally preferred. The 20 L-amino acids commonly found in proteins areidentified herein by the conventional three-letter or one-letterabbreviations.

A modulating agent may also contain rare amino acids (such as4-hydroxyproline or hydroxylysine), organic acids or amides and/orderivatives of common amino acids, such as amino acids having theC-terminal carboxylate esterified (e.g., benzyl, methyl or ethyl ester)or amidated and/or having modifications of the N-terminal amino group(e.g., acetylation or alkoxycarbonylation), with or without any of awide variety of side-chain modifications and/or substitutions (e.g.,methylation, benzylation, t-butylation, tosylation, alkoxycarbonylation,and the like). Preferred derivatives include amino acids having aC-terminal amide group. Residues other than common amino acids that maybe present with a modulating agent include, but are not limited to,2-mercaptoaniline, 2-mercaptoproline, ornithine, diaminobutyric acid,α-aminoadipic acid, m-aminomethylbenzoic acid and α,β-diaminopropionicacid.

Peptide modulating agents (and peptide portions of modulating agents) asdescribed herein may be synthesized by methods well known in the art,including chemical synthesis and recombinant DNA methods. For modulatingagents up to about 50 residues in length, chemical synthesis may beperformed using solid phase peptide synthesis techniques, in which apeptide linkage occurs through the direct condensation of the α-aminogroup of one amino acid with the α-carboxy group of the other amino acidwith the elimination of a water molecule. Peptide bond synthesis bydirect condensation, as formulated above, requires suppression of thereactive character of the amino group of the first and of the carboxylgroup of the second amino acid. The masking substitutents must permittheir ready removal, without inducing breakdown of the labile peptidemolecule.

Solid phase peptide synthesis uses an insoluble polymer for supportduring organic synthesis. The polymer-supported peptide chain permitsthe use of simple washing and filtration steps instead of laboriouspurifications at intermediate steps. Solid-phase peptide synthesis maygenerally be performed according to the method of Merrifield et al., J.Am. Chem. Soc. 85:2149, 1963, which involves assembling a linear peptidechain on a resin support using protected amino acids. Solid phasepeptide synthesis typically utilizes either the Boc or Fmoc strategy.The Boc strategy uses a 1% cross-linked polystyrene resin. The standardprotecting group for α-amino functions is the tert-butyloxycarbonyl(Boc) group. This group can be removed with dilute solutions of strongacids such as 25% trifluoroacetic acid (TFA). The next Boc-amino acid istypically coupled to the amino acyl resin using dicyclohexylcarbodiimide(DCC). Following completion of the assembly, the peptide-resin istreated with anhydrous HF to cleave the benzyl ester link and liberatethe free peptide. Side-chain functional groups are usually blockedduring synthesis by benzyl-derived blocking groups, which are alsocleaved by HF. The free peptide is then extracted from the resin with asuitable solvent, purified and characterized. Newly synthesized peptidescan be purified, for example, by gel filtration, HPLC, partitionchromatography and/or ion-exchange chromatography, and may becharacterized by, for example, mass spectrometry or amino acid sequenceanalysis. In the Boc strategy, C-terminal amidated peptides can beobtained using benzhydrylamine or methylbenzhydrylamine resins, whichyield peptide amides directly upon cleavage with HF.

In the procedures discussed above, the selectivity of the side-chainblocking groups and of the peptide-resin link depends upon thedifferences in the rate of acidolytic cleavage. Orthoganol systems havebeen introduced in which the side-chain blocking groups and thepeptide-resin link are completely stable to the reagent used to removethe α-protecting group at each step of the synthesis. The most common ofthese methods involves the 9-fluorenylmethyloxycarbonyl (Fmoc) approach.Within this method, the side-chain protecting groups and thepeptide-resin link are completely stable to the secondary amines usedfor cleaving the N-α-Fmoc group. The side-chain protection and thepeptide-resin link are cleaved by mild acidolysis. The repeated contactwith base makes the Merrifield resin unsuitable for Fmoc chemistry, andp-alkoxybenzyl esters linked to the resin are generally used.Deprotection and cleavage are generally accomplished using TFA.

Those of ordinary skill in the art will recognize that, in solid phasesynthesis, deprotection and coupling reactions must go to completion andthe side-chain blocking groups must be stable throughout the entiresynthesis. In addition, solid phase synthesis is generally most suitablewhen peptides are to be made on a small scale.

Acetylation of the N-terminus can be accomplished by reacting the finalpeptide with acetic anhydride before cleavage from the resin.C-amidation is accomplished using an appropriate resin such asmethylbenzhydrylamine resin using the Boc technology.

Following synthesis of a linear peptide, with or without N-acetylationand/or C-amidation, cyclization may be achieved if desired by any of avariety of techniques well known in the art. Within one embodiment, abond may be generated between reactive amino acid side chains. Forexample, a disulfide bridge may be formed from a linear peptidecomprising two thiol-containing residues by oxidizing the peptide usingany of a variety of methods. Within one such method, air oxidation ofthiols can generate disulfide linkages over a period of several daysusing either basic or neutral aqueous media. The peptide is used in highdilution to minimize aggregation and intermolecular side reactions. Thismethod suffers from the disadvantage of being slow but has the advantageof only producing H₂O as a side product. Alternatively, strong oxidizingagents such as I₂ and K₃Fe(CN)₆ can be used to form disulfide linkages.Those of ordinary skill in the art will recognize that care must betaken not to oxidize the sensitive side chains of Met, Tyr, Trp or His.Cyclic peptides produced by this method require purification usingstandard techniques, but this oxidation is applicable at acid pHs.Oxidizing agents also allow concurrent deprotection/oxidation ofsuitable S-protected linear precursors to avoid premature, nonspecificoxidation of free cysteine.

DMSO, unlike I₂ and K₃Fe(CN)₆, is a mild oxidizing agent which does notcause oxidative side reactions of the nucleophilic amino acids mentionedabove. DMSO is miscible with H₂O at all concentrations, and oxidationscan be performed at acidic to neutral pHs with harmless byproducts.Methyltrichlorosilane-diphenylsulfoxide may alternatively be used as anoxidizing agent, for concurrent deprotection/oxidation of S-Acm, S-Tacmor S-t-Bu of cysteine without affecting other nucleophilic amino acids.There are no polymeric products resulting from intermolecular disulfidebond formation. Suitable thiol-containing residues for use in suchoxidation methods include, but are not limited to, cysteine,β,β-dimethyl cysteine (penicillamine or Pen), β,β-tetramethylenecysteine (Tmc), β,β-pentamethylene cysteine (Pmc), β-mercaptopropionicacid (Mpr), β,β-pentamethylene-β-mercaptopropionic acid (Pmp),2-mercaptobenzene, 2-mercaptoaniline and 2-mercaptoproline.

Within another embodiment, cyclization may be achieved by amide bondformation. For example, a peptide bond may be formed between terminalfunctional groups (i.e., the amino and carboxy termini of a linearpeptide prior to cyclization). Within another such embodiment, thelinear peptide comprises a D-amino acid. Alternatively, cyclization maybe accomplished by linking one terminus and a residue side chain orusing two side chains, with or without an N-terminal acetyl group and/ora C-terminal amide. Residues capable of forming a lactam bond includelysine, ornithine (Orn), α-amino adipic acid, m-aminomethylbenzoic acid,α,β-diaminopropionic acid, glutamate or aspartate.

Methods for forming amide bonds are well known in the art and are basedon well established principles of chemical reactivity. Within one suchmethod, carbodiimide-mediated lactam formation can be accomplished byreaction of the carboxylic acid with DCC, DIC, EDAC or DCCI, resultingin the formation of an O-acylurea that can be reacted immediately withthe free amino group to complete the cyclization. The formation of theinactive N-acylurea, resulting from O→N migration, can be circumventedby converting the O-acylurea to an active ester by reaction with anN-hydroxy compound such as 1-hydroxybenzotriazole, 1-hydroxysuccinimide,1-hydroxynorbornene carboxamide or ethyl 2-hydroximino-2-cyanoacetate.In addition to minimizing O→N migration, these additives also serve ascatalysts during cyclization and assist in lowering racemization.Alternatively, cyclization can be performed using the azide method, inwhich a reactive azide intermediate is generated from an alkyl ester viaa hydrazide. Hydrazinolysis of the terminal ester necessitates the useof a t-butyl group for the protection of side chain carboxyl functionsin the acylating component. This limitation can be overcome by usingdiphenylphosphoryl acid (DPPA), which furnishes an azide directly uponreaction with a carboxyl group. The slow reactivity of azides and theformation of isocyanates by their disproportionation restrict theusefulness of this method. The mixed anhydride method of lactamformation is widely used because of the facile removal of reactionby-products. The anhydride is formed upon reaction of the carboxylateanion with an alkyl chloroformate or pivaloyl chloride. The attack ofthe amino component is then guided to the carbonyl carbon of theacylating component by the electron donating effect of the alkoxy groupor by the steric bulk of the pivaloyl chloride t-butyl group, whichobstructs attack on the wrong carbonyl group. Mixed anhydrides withphosphoric acid derivatives have also been successfully used.Alternatively, cyclization can be accomplished using activated esters.The presence of electron withdrawing substitutents on the alkoxy carbonof esters increases their susceptibility to aminolysis. The highreactivity of esters of p-nitrophenol, N-hydroxy compounds andpolyhalogenated phenols has made these “active esters” useful in thesynthesis of amide bonds. The last few years have witnessed thedevelopment of benzotriazolyloxytris-(dimethylamino)phosphoniumhexafluorophosphonate (BOP) and its congeners as advantageous couplingreagents. Their performance is generally superior to that of the wellestablished carbodiimide amide bond formation reactions.

Within a further embodiment, a thioether linkage may be formed betweenthe side chain of a thiol-containing residue and an appropriatelyderivatized α-amino acid. By way of example, a lysine side chain can becoupled to bromoacetic acid through the carbodiimide coupling method(DCC, EDAC) and then reacted with the side chain of any of the thiolcontaining residues mentioned above to form a thioether linkage. Inorder to form dithioethers, any two thiol containing side-chains can bereacted with dibromoethane and diisopropylamine in DMF.

For longer modulating agents, recombinant methods are preferred forsynthesis. Within such methods, all or part of a modulating agent can besynthesized in living cells, using any of a variety of expressionvectors known to those of ordinary skill in the art to be appropriatefor the particular host cell. Suitable host cells may include bacteria,yeast cells, mammalian cells, insect cells, plant cells, algae and otheranimal cells (e.g., hybridoma, CHO, myeloma). The DNA sequencesexpressed in this manner may encode portions of an endogenous cadherinor other adhesion molecule. Such sequences may be prepared based onknown cDNA or genomic sequences (see Blaschuk et al., J. Mol. Biol.211:679-682, 1990), or from sequences isolated by screening anappropriate library with probes designed based on the sequences of knowncadherins. Such screens may generally be performed as described inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratories, Cold Spring Harbor, N.Y., 1989 (and referencescited therein). Polymerase chain reaction (PCR) may also be employed,using oligonucleotide primers in methods well known in the art, toisolate nucleic acid molecules encoding all or a portion of anendogenous adhesion molecule. To generate a nucleic acid moleculeencoding a desired modulating agent, an endogenous cadherin sequence maybe modified using well known techniques. For example, portions encodingone or more CAR sequences may be joined, with or without separation bynucleic acid regions encoding linkers, as discussed above.Alternatively, portions of the desired nucleic acid sequences may besynthesized using well known techniques, and then ligated together toform a sequence encoding the modulating agent.

As noted above, instead of (or in addition to) an HAV-BM sequence, amodulating agent may comprise an antibody, or antigen-binding fragmentthereof, that specifically binds to an HAV-BM sequence. As used herein,an antibody, or antigen-binding fragment thereof, is said to“specifically bind” to an HAV-BM sequence (with or without flankingamino acids) if it reacts at a detectable level with a peptidecontaining that sequence, and does not react detectably with peptidescontaining a different CAR sequence or a sequence in which the order ofamino acid residues in the cadherin CAR sequence and/or flankingsequence is altered. Such antibody binding properties may be assessedusing an ELISA, as described by Newton et al., Develop. Dynamics197:1-13, 1993.

Polyclonal and monoclonal antibodies may be raised against an HAV-BMsequence using conventional techniques. See, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Inone such technique, an immunogen comprising the HAV-BM sequence isinitially injected into any of a wide variety of mammals (e.g., mice,rats, rabbits, sheep or goats). The smaller immunogens (i.e., less thanabout 20 amino acids) should be joined to a carrier protein, such asbovine serum albumin or keyhole limpet hemocyanin. Following one or moreinjections, the animals are bled periodically. Polyclonal antibodiesspecific for the CAR sequence may then be purified from such antiseraby, for example, affinity chromatography using the modulating agent orantigenic portion thereof coupled to a suitable solid support.

Monoclonal antibodies specific for the HAV-BM sequence may be prepared,for example, using the technique of Kohler and Milstein, Eur. J.Immunol. 6:511-519, 1976, and improvements thereto. Briefly, thesemethods involve the preparation of immortal cell lines capable ofproducing antibodies having the desired specificity from spleen cellsobtained from an animal immunized as described above. The spleen cellsare immortalized by, for example, fusion with a myeloma cell fusionpartner, preferably one that is syngeneic with the immunized animal.Single colonies are selected and their culture supernatants tested forbinding activity against the modulating agent or antigenic portionthereof. Hybridomas having high reactivity and specificity arepreferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies, with or without the use of various techniques knownin the art to enhance the yield. Contaminants may be removed from theantibodies by conventional techniques, such as chromatography, gelfiltration, precipitation and extraction. Antibodies having the desiredactivity may generally be identified using immunofluorescence analysesof tissue sections, cell or other samples where the target cadherin islocalized.

Within preferred embodiments, such monoclonal antibodies are specificfor particular cadherins (e.g., the antibodies bind to E-cadherin, butdo not bind significantly to N-cadherin, or vise versa). Such antibodiesmay be prepared as described above, using an immunogen that comprises(in addition to a minimal HAV-BM sequence) sufficient flanking sequenceto generate the desired specificity. To evaluate the specificity of aparticular antibody, representative assays as described herein and/orconventional antigen-binding assays may be employed. Such antibodies maygenerally be used for therapeutic, diagnostic and assay purposes, asdescribed herein. For example, such antibodies may be linked to a drugand administered to a mammal to target the drug to a particularcadherin-expressing cell.

Within certain embodiments, the use of antigen-binding fragments ofantibodies may be preferred. Such fragments include Fab fragments, whichmay be prepared using standard techniques. Briefly, immunoglobulins maybe purified from rabbit serum by affinity chromatography on Protein Abead columns (Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; see especially page 309) and digested bypapain to yield Fab and Fc fragments. The Fab and Fc fragments may beseparated by affinity chromatography on protein A bead columns (Harlowand Lane, 1988, pages 628-29).

Within certain embodiments, antibodies may be used within methods inwhich enhanced cell adhesion is desired, as described above. Forexample, antibodies may be used within the above methods for enhancingand/or directing neurite outgrowth in vitro or in vivo. Antibodies maybe used within the lumen of a tubular nerve guide or may be attached toa fiber nerve guide, suture or other solid support and used as describedabove for peptide modulating agents. Antibody dosages are sufficient toenhance or direct neurite outgrowth, and will vary with the method ofadministration and the condition to be treated.

Antibodies may also be used as a “biological glue,” as described aboveto bind multiple cadherin-expressing cells within a variety of contexts,such as to enhance wound healing and/or reduce scar tissue, and/or tofacilitate cell adhesion in skin grafting or prosthetic implants. Ingeneral, the amount of matrix-linked antibody administered to a wound,graft or implant site varies with the severity of the wound and/or thenature of the wound, graft, or implant, but may vary as discussed above.Antibodies may also be linked to any of a variety of support materials,as described above, for use in tissue culture or bioreactors.

Antibodies (or, preferably, antigen-binding fragments thereof) may alsobe used in situations where inhibition of cell adhesion is desired. Suchantibodies or fragments may be used, for example, for treatment ofdemyelinating diseases, such as MS, or to inhibit interactions betweentumor cells, as described above. The use of Fab fragments is generallypreferred.

Evaluation of Modulating Agent Activity

As noted above, native HAV-BM sequences, as well as analogues andmimetics thereof, bind to a classical cadherin, preferably within ornear an HAV sequence, and modulate a cadherin-mediated response. Theability to bind to a cadherin sequence may generally be evaluated usingany binding assay known to those of ordinary skill in the art. Forexample, a Pharmacia Biosensor machine may be used, as discussed inJonsson et al., Biotechniques 11:520-27, 1991. A specific example of thetechnology that measures the interaction of peptides with molecules canbe found in Williams et al., J. Biol. Chem. 272:8539-8545, 1997.Real-time BIA (Biomolecular Interaction Analysis) uses the opticalphenomenon surface plasmon resonance to monitor biomolecularinteractions. The detection depends upon changes in the massconcentration of macromolecules at the biospecific interface, which inturn depends upon the immobilization of test molecule or peptide(referred to as the ligand) to the surface of a Biosensor chip, followedby binding of the interacting molecule (referred to as the analyte) tothe ligand. Binding to the chip is measured in real-time in arbitraryunits of resonance (RU).

For example, surface plasmon resonance experiments may be carried outusing a BIAcore X™ Biosensor (Pharmacia Ltd., BIAcore, Uppsala, Sweden).Parallel flow cells of CM 5 sensor chips may be derivatized, using theamine coupling method, with streptavidin (200 μg/ml) in 10 mM SodiumAcetate, pH 4.0, according to the manufacturer's protocol. Approximately2100-2600 resonance units (RU) of ligand may be immobilized,corresponding to a concentration of about 2.1-2.6 ng/mm². The chips maythen coated be with a peptide comprising a known or putative HAV-BM, oranalogue or mimetic thereof. Any non-specifically bound peptide isremoved.

To determine binding, test analytes (e.g., cadherin peptides, such asHAV-containing peptides) may be placed in running buffer and passedsimultaneously over test and control flow cells. After a period of freebuffer flow, any analyte remaining bound to the surface may be removedwith, for example, a pulse of 0.1% SDS bringing the signal back tobaseline. Specific binding to the derivatized sensor chips may bedetermined automatically by the system by subtraction of test fromcontrol flow cell responses. In general, an HAV-BM, or a mimetic oranalogue thereof, binds an HAV-containing peptide at a detectable levelwithin such as assay. Preferably, the level of binding is at least thatobserved for a native HAV-BM as provided herein under similarconditions.

The ability to modulate a cadherin-mediated function may be evaluatedusing any of a variety of in vitro assays designed to measure the effectof the peptide on a typical cadherin response. As noted above,modulating agents may be capable of enhancing or inhibiting acadherin-mediated function. The ability of an agent to modulate celladhesion may generally be evaluated in vitro by assaying the effect onone or more of the following: (1) neurite outgrowth, (2) Schwanncell-astrocyte adhesion, (3) Schwann cell migration on astrocytemonolayers, (4) adhesion between endothelial cells, (5) adhesion betweenepithelial cells (e.g., normal rat kidney cells and/or human skin)and/or (6) adhesion between cancer cells. In general, a modulating agentis an inhibitor of cell adhesion if, within one or more of theserepresentative assays, contact of the test cells with the modulatingagent results in a discernible disruption of cell adhesion. Modulatingagents that enhance cell adhesion (e.g., agents comprising multipleHAV-BM sequences and/or linked to a support material) are considered tobe modulators of cell adhesion if they are capable of enhancing neuriteoutgrowth as described below or are capable of promoting cell adhesion,as judged by plating assays to assess epithelial cell adhesion to amodulating agent attached to a support material, such as tissue cultureplastic.

Within a representative neurite outgrowth assay, neurons may be culturedon a monolayer of cells (e.g., 3T3 fibroblasts) that express N-cadherin.Neurons grown on such cells (under suitable conditions and for asufficient period of time) extend neurites that are typically, onaverage, twice as long as neurites extended from neurons cultured on 3T3cells that do not express N-cadherin. For example, neurons may becultured on monolayers of 3T3 cells transfected with cDNA encodingN-cadherin essentially as described by Doherty and Walsh, Curr. Op.Neurobiol. 4:49-55, 1994; Williams et al., Neuron 13:583-594, 1994; Hallet al., Cell Adhesion and Commun. 3:441-450, 1996; Doherty and Walsh,Mol. Cell. Neurosci. 8:99-111, 1994; and Safell et al., Neuron18:231-242, 1997. Briefly, monolayers of control 3T3 fibroblasts and 3T3fibroblasts that express N-cadherin may be established by overnightculture of 80,000 cells in individual wells of an 8-chamber well tissueculture slide. 3000 cerebellar neurons isolated from post-natal day 3mouse brains may be cultured for 18 hours on the various monolayers incontrol media (SATO/2% FCS), or media supplemented with variousconcentrations of the modulating agent or control peptide. The culturesmay then be fixed and stained for GAP43 which specifically binds to theneurons and their neurites. The length of the longest neurite on eachGAP43 positive neuron may be measured by computer assisted morphometry.

A modulating agent that modulates N-cadherin-mediated cell adhesion mayinhibit or enhance such neurite outgrowth. Under the conditionsdescribed above, the presence of 500 μg/mL of a modulating agent thatdisrupts neural cell adhesion should result in a decrease in the meanneurite length by at least 50%, relative to the length in the absence ofmodulating agent or in the presence of a negative control peptide.Alternatively, the presence of 500 μg/mL of a modulating agent thatenhances neural cell adhesion should result in an increase in the meanneurite length by at least 50%.

The effect of a modulating agent on Schwann cell adhesion to astrocytesmay generally be evaluated using a cell adhesion assay. Briefly, Schwanncells fluorescently labeled with Di-I may be plated onto an astrocyticsurface (e.g., a glass coverslip coated with a monolayer of astrocytes)and incubated on a shaking platform (e.g., 25 rpm for 30 minutes) in thepresence and absence of modulating agent at a concentration ofapproximately 1 mg/mL. Cells may then be washed (e.g., in Hanks medium)to remove non-attached cells. The attached cells may then be fixed andcounted (e.g., using a fluorescent microscope). In general, 1 mg/mL of amodulating agent results in an increase or decrease in cell adhesion ofat least 50%. This assay evaluates the effect of a modulating agent onN-cadherin mediated cell adhesion.

Schwann cell migration may generally be evaluated using amicro-inverted-coverslip assay. In this assay, a dense Schwann cellculture is established on coverslip fragments and Schwann cell migrationaway from the fragment edge is measured. Briefly, Schwann cellsfluorescently labeled with Di-I may be plated on polylysine- andlaminin-coated fragments of a glass coverslip and allowed to bind to thesurface for 16-18 hours. Cells may then be washed (e.g., in Hanksmedium) to remove non-attached cells, and then inverted, with cellsfacing downward onto an astrocyte-coated surface. Cultures are thenincubated further for 2 days in the presence or absence of modulatingagent at a concentration of approximately 1 mg/mL and fixed. The maximummigration distance from the edge of the coverslip fragment may then bemeasured. At a level of 1 mg/mL, a modulating agent results in anincrease or decrease in the maximum migration distance of at least 50%.This assay evaluates the effect of a modulating agent on N-cadherinmediated cell adhesion.

Within certain cell adhesion assays, the addition of a modulating agentto cells that express a cadherin results in disruption of cell adhesion.A “cadherin-expressing cell,” as used herein, may be any type of cellthat expresses at least one cadherin on the cell surface at a detectablelevel, using standard techniques such as immunocytochemical protocols(e.g., Blaschuk and Farookhi, Dev. Biol. 136:564-567, 1989).Cadherin-expressing cells include endothelial, epithelial and/or cancercells. For example, such cells may be plated under standard conditionsthat, in the absence of modulating agent, permit cell adhesion. In thepresence of modulating agent (e.g., 500 μg/mL), disruption of celladhesion may be determined visually within 24 hours, by observingretraction of the cells from one another.

For use within one such assay, bovine pulmonary artery endothelial cellsmay be harvested by sterile ablation and digestion in 0.1% collagenase(type II; Worthington Enzymes, Freehold, N.J.). Cells may be maintainedin Dulbecco's minimum essential medium supplemented with 10% fetal calfserum and 1% antibiotic-antimycotic at 37° C. in 7% CO₂ in air. Culturesmay be passaged weekly in trypsin-EDTA and seeded onto tissue cultureplastic at 20,000 cells/cm². Endothelial cultures may be used at 1 weekin culture, which is approximately 3 days after culture confluency isestablished. The cells may be seeded onto coverslips and treated (e.g.,for 30 minutes) with modulating agent or a control compound at, forexample, 500 μg/ml and then fixed with 1% paraformaldehyde. As notedabove, disruption of cell adhesion may be determined visually within 24hours, by observing retraction of the cells from one another. This assayevaluates the effect of a modulating agent on N-cadherin mediated celladhesion.

Within another such assay, the effect of a modulating agent on normalrat kidney (NRK) cells may be evaluated. According to a representativeprocedure, NRK cells (ATCC #1571-CRL) may be plated at 10-20,000 cellsper 35 mm tissue culture flasks containing DMEM with 10% FCS andsub-cultured periodically (Laird et al., J. Cell Biol. 131:1193-1203,1995). Cells may be harvested and replated in 35 mm tissue cultureflasks containing 1 mm coverslips and incubated until 50-65% confluent(24-36 hours). At this time, coverslips may be transferred to a 24-wellplate, washed once with fresh DMEM and exposed to modulating agent at aconcentration of, for example, 1 mg/mL for 24 hours. Fresh modulatingagent may then be added, and the cells left for an additional 24 hours.Cells may be fixed with 100% methanol for 10 minutes and then washedthree times with PBS. Coverslips may be blocked for 1 hour in 2% BSA/PBSand incubated for a further 1 hour in the presence of mouseanti-E-cadherin antibody (Transduction Labs, 1:250 dilution). Primaryand secondary antibodies may be diluted in 2% BSA/PBS. Followingincubation in the primary antibody, coverslips may be washed three timesfor 5 minutes each in PBS and incubated for 1 hour with donkeyanti-mouse antibody conjugated to fluorescein (diluted 1:200). Followingfurther washes in PBS (3×5 min) coverslips can be mounted and viewed byconfocal microscopy.

In the absence of modulating agent, NRK cells form characteristictightly adherent monolayers with a cobblestone morphology in which cellsdisplay a polygonal shape. NRK cells that are treated with a modulatingagent that disrupts E-cadherin mediated cell adhesion may assume anon-polygonal and elongated morphology (i.e., a fibroblast-like shape)within 48 hours of treatment with 1 mg/mL of modulating agent. Gapsappear in confluent cultures of such cells. In addition, 1 mg/mL of sucha modulating agent reproducibly induces a readily apparent reduction incell surface staining of E-cadherin, as judged by immunofluorescencemicroscopy (Laird et al., J. Cell Biol. 131:1193-1203, 1995), of atleast 75% within 48 hours.

A third cell adhesion assay involves evaluating the effect of amodulating agent on permeability of adherent epithelial and/orendothelial cell layers. For example, the effect of permeability onhuman skin may be evaluated. Such skin may be derived from a naturalsource or may be synthetic. Human abdominal skin for use in such assaysmay generally be obtained from humans at autopsy within 24 hours ofdeath. Briefly, a modulating agent (e.g., 500 μg/ml) and a test marker(e.g., the fluorescent markers Oregon Green™ and Rhodamine Green™Dextran) may be dissolved in a sterile buffer (e.g., phosphate buffer,pH 7.2), and the ability of the marker to penetrate through the skin andinto a receptor fluid (e.g., phosphate buffer) may be measured using aFranz Cell apparatus (Franz, Curr. Prob. Dermatol. 7:58-68, 1978; Franz,J. Invest. Dermatol. 64:190-195, 1975). The penetration of the markersthrough the skin may be assessed at, for example, 6, 12, 24, 36, and 48hours after the start of the experiment. In general, a modulating agentthat enhances the permeability of human skin results in a statisticallysignificant increase in the amount of marker in the receptor compartmentafter 6-48 hours in the presence of 500 μg/mL modulating agent. Thisassay evaluates the effect of a modulating agent on E-cadherin mediatedcell adhesion.

Modulating Agent Modification and Formulations

A modulating agent as described herein may, but need not, be linked toone or more additional molecules. In particular, as discussed below, itmay be beneficial for certain applications to link multiple modulatingagents (which may, but need not, be identical) to a support material,such as a support molecule (e.g., keyhole limpet hemocyanin) or a solidsupport, such as a polymeric matrix (which may be formulated as amembrane or microstructure, such as an ultra thin film), a containersurface (e.g. the surface of a tissue culture plate or the interiorsurface of a bioreactor), or a bead or other particle, which may beprepared from a variety of materials including glass, plastic orceramics. For certain applications, biodegradable support materials arepreferred, such as cellulose and derivatives thereof, collagen, spidersilk or any of a variety of polyesters (e.g., those derived from hydroxyacids and/or lactones) or sutures (see U.S. Pat. No. 5,245,012). Withincertain embodiments, modulating agents and molecules comprising otherCAR sequence(s) (e.g., HAV, RGD or LYHY (SEQ ID NO:70)) may be attachedto a support such as a polymeric matrix, preferably in an alternatingpattern.

Suitable methods for linking a modulating agent to a support materialwill depend upon the composition of the support and the intended use,and will be readily apparent to those of ordinary skill in the art.Attachment may generally be achieved through noncovalent association,such as adsorption or affinity or, preferably, via covalent attachment(which may be a direct linkage between a modulating agent and functionalgroups on the support, or may be a linkage by way of a cross-linkingagent). Attachment of a modulating agent by adsorption may be achievedby contact, in a suitable buffer, with a solid support for a suitableamount of time. The contact time varies with temperature, but isgenerally between about 5 seconds and 1 day, and typically between about10 seconds and 1 hour.

Covalent attachment of a modulating agent to a molecule or solid supportmay generally be achieved by first reacting the support material with abifunctional reagent that will also react with a functional group, suchas a hydroxyl or amino group, on the modulating agent. For example, amodulating agent may be bound to an appropriate polymeric support orcoating using benzoquinone, by condensation of an aldehyde group on thesupport with an amine and an active hydrogen on the modulating agent orby condensation of an amino group on the support with a carboxylic acidon the modulating agent. A preferred method of generating a linkage isvia amino groups using glutaraldehyde. A modulating agent may be linkedto cellulose via ester linkages. Similarly, amide linkages may besuitable for linkage to other molecules such as keyhole limpethemocyanin or other support materials. Multiple modulating agents and/ormolecules comprising other CAR sequences may be attached, for example,by random coupling, in which equimolar amounts of such molecules aremixed with a matrix support and allowed to couple at random.

Although modulating agents as described herein may preferentially bindto specific tissues or cells, and thus may be sufficient to target adesired site in vivo, it may be beneficial for certain applications toinclude an additional targeting agent. Accordingly, a targeting agentmay also, or alternatively, be linked to a modulating agent tofacilitate targeting to one or more specific tissues. As used herein, a“targeting agent,” may be any substance (such as a compound or cell)that, when linked to a modulating agent enhances the transport of themodulating agent to a target tissue, thereby increasing the localconcentration of the modulating agent. Targeting agents includeantibodies or fragments thereof, receptors, ligands and other moleculesthat bind to cells of, or in the vicinity of, the target tissue. Knowntargeting agents include serum hormones, antibodies against cell surfaceantigens, lectins, adhesion molecules, tumor cell surface bindingligands, steroids, cholesterol, lymphokines, fibrinolytic enzymes andthose drugs and proteins that bind to a desired target site. Among themany monoclonal antibodies that may serve as targeting agents areanti-TAC, or other interleukin-2 receptor antibodies; 9.2.27 andNR-ML-05, reactive with the 250 kilodalton human melanoma-associatedproteoglycan; and NR-LU-10, reactive with a pancarcinoma glycoprotein.An antibody targeting agent may be an intact (whole) molecule, afragment thereof, or a functional equivalent thereof. Examples ofantibody fragments are F(ab′)2, -Fab′, Fab and F[v] fragments, which maybe produced by conventional methods or by genetic or proteinengineering. Linkage is generally covalent and may be achieved by, forexample, direct condensation or other reactions, or by way of bi- ormulti-functional linkers. Within other embodiments, it may also bepossible to target a polynucleotide encoding a modulating agent to atarget tissue, thereby increasing the local concentration of modulatingagent. Such targeting may be achieved using well known techniques,including retroviral and adenoviral infection.

For certain embodiments, it may be beneficial to also, or alternatively,link a drug to a modulating agent. As used herein, the term “drug”refers to any bioactive agent intended for administration to a mammal toprevent or treat a disease or other undesirable condition. Drugs includehormones, growth factors, proteins, peptides and other compounds. Theuse of certain specific drugs within the context of the presentinvention is discussed below.

Within certain aspects of the present invention, one or more modulatingagents as described herein may be present within a pharmaceuticalcomposition. A pharmaceutical composition comprises one or moremodulating agents in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers (e.g., neutral buffered saline orphosphate buffered saline), carbohydrates (e.g., glucose, mannose,sucrose or dextrans), mannitol, proteins, polypeptides or amino acidssuch as glycine, antioxidants, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives.Within yet other embodiments, compositions of the present invention maybe formulated as a lyophilizate. One or more modulating agents (alone orin combination with a targeting agent and/or drug) may, but need not, beencapsulated within liposomes using well known technology. Compositionsof the present invention may be formulated for any appropriate manner ofadministration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous, orintramuscular administration.

For certain embodiments, as discussed below, a pharmaceuticalcomposition may further comprise a modulator of cell adhesion that ismediated by one or more molecules other than cadherins. Such modulatorsmay generally be prepared as described above, incorporating one or morenon-cadherin CAR sequences and/or antibodies thereto in place of theHAV-BM sequences and antibodies. Such compositions are particularlyuseful for situations in which it is desirable to inhibit cell adhesionmediated by multiple cell-adhesion molecules, such as other members ofthe cadherin gene superfamily that are not classical cadherins (e.g.,Dsg and Dsc); integrins; members of the immunoglobulin supergene family,such as N-CAM; and other uncategorized transmembrane proteins, such asoccludin, as well as extracellular matrix proteins such as laminin,fibronectin, collagens, vitronectin, entactin and tenascin. PreferredCAR sequences for use within such a modulator include HAV, RGD, YIGSR(SEQ ID NO:66), KYSFNYDGSE (SEQ ID NO:67), a Dsc or Dsg CAR sequence, aclaudin CAR sequence, a JAM CAR sequence and/or LYHY (SEQ ID NO:70).

A pharmaceutical composition may also, or alternatively, contain one ormore drugs, which may be linked to a modulating agent or may be freewithin the composition. Virtually any drug may be administered incombination with a modulating agent as described herein, for a varietyof purposes as described below. Examples of types of drugs that may beadministered with a modulating agent include analgesics, anesthetics,antianginals, antifungals, antibiotics, anticancer drugs (e.g., taxol ormitomycin C), antiinflammatories (e.g., ibuprofen and indomethacin),anthelmintics, antidepressants, antidotes, antiemetics, antihistamines,antihypertensives, antimalarials, antimicrotubule agents (e.g.,colchicine or vinca alkaloids), antimigraine agents, antimicrobials,antiphsychotics, antipyretics, antiseptics, anti-signaling agents (e.g.,protein kinase C inhibitors or inhibitors of intracellular calciummobilization), antiarthritics, antithrombin agents, antituberculotics,antitussives, antivirals, appetite suppressants, cardioactive drugs,chemical dependency drugs, cathartics, chemotherapeutic agents,coronary, cerebral or peripheral vasodilators, contraceptive agents,depressants, diuretics, expectorants, growth factors, hormonal agents,hypnotics, immunosuppression agents, narcotic antagonists,parasympathomimetics, sedatives, stimulants, sympathomimetics, toxins(e.g., cholera toxin), tranquilizers and urinary antiinfectives.

For imaging purposes, any of a variety of diagnostic agents may beincorporated into a pharmaceutical composition, either linked to amodulating agent or free within the composition. Diagnostic agentsinclude any substance administered to illuminate a physiologicalfunction within a patient, while leaving other physiological functionsgenerally unaffected. Diagnostic agents include metals, radioactiveisotopes and radioopaque agents (e.g., gallium, technetium, indium,strontium, iodine, barium, bromine and phosphorus-containing compounds),radiolucent agents, contrast agents, dyes (e.g., fluorescent dyes andchromophores) and enzymes that catalyze a calorimetric or fluorometricreaction. In general, such agents may be attached using a variety oftechniques as described above, and may be present in any orientation.

The compositions described herein may be administered as part of asustained release formulation (i.e., a formulation such as a capsule orsponge that effects a slow release of modulating agent followingadministration). Such formulations may generally be prepared using wellknown technology and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Sustained-release formulations may contain a modulating agentdispersed in a carrier matrix and/or contained within a reservoirsurrounded by a rate controlling membrane (see, e.g. European PatentApplication 710,491 A). Carriers for use within such formulations arebiocompatible, and may also be biodegradable; preferably the formulationprovides a relatively constant level of modulating agent release. Theamount of modulating agent contained within a sustained releaseformulation depends upon the site of implantation, the rate and expectedduration of release and the nature of the condition to be treated orprevented.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented).Appropriate dosages and a suitable duration and frequency ofadministration will be determined by such factors as the condition ofthe patient, the type and severity of the patient's disease and themethod of administration. In general, an appropriate dosage andtreatment regimen provides the modulating agent(s) in an amountsufficient to provide therapeutic and/or prophylactic benefit. Withinparticularly preferred embodiments of the invention, a modulating agentor pharmaceutical composition as described herein may be administered ata dosage ranging from 0.001 to 50 mg/kg body weight, preferably from 0.1to 20 mg/kg, on a regimen of single or multiple daily doses. For topicaladministration, a cream typically comprises an amount of modulatingagent ranging from 0.00001% to 1%, preferably 0.0001% to 0.002%. Fluidcompositions typically contain about 10 ng/ml to 5 mg/ml, preferablyfrom about 10 μg to 2 mg/mL modulating agent. Appropriate dosages maygenerally be determined using experimental models and/or clinicaltrials. In general, the use of the minimum dosage that is sufficient toprovide effective therapy is preferred. Patients may generally bemonitored for therapeutic effectiveness using assays suitable for thecondition being treated or prevented, which will be familiar to those ofordinary skill in the art.

Therapeutic Methods Employing Modulating Agents

In general, the modulating agents and compositions described herein maybe used for modulating the adhesion of cadherin-expressing cells (i.e.,cells that express one or more of E-cadherin, N-cadherin, P-cadherin,R-cadherin and/or other cadherin(s) containing the HAV-BM sequence,including as yet undiscovered cadherins). Such modulation may beperformed in vitro and/or in vivo, preferably in a mammal such as ahuman. As noted above, modulating agents for purposes that involve thedisruption of cadherin-mediated cell adhesion may comprise an HAV-BMsequence, multiple HAV-BM sequences in close proximity and/or anantibody (or an antigen-binding fragment thereof) that recognizes anHAV-BM sequence. When it is desirable to also disrupt cell adhesionmediated by other adhesion molecules, a modulating agent mayadditionally comprise one or more CAR sequences bound by such adhesionmolecules (and/or antibodies or fragments thereof that bind suchsequences), preferably separated from each other and from the HAV-BMsequence by linkers. As noted above, such linkers may or may notcomprise one or more amino acids. For enhancing cell adhesion, amodulating agent may contain multiple HAV-BM sequences or antibodies (orfragments), preferably separated by linkers, and/or may be linked to asingle molecule or to a support material as described above.

Certain methods involving the disruption of cell adhesion as describedherein have an advantage over prior techniques in that they permit thepassage of molecules that are large and/or charged across barriers ofcadherin-expressing cells. As described in greater detail below,modulating agents as described herein may also be used to disrupt orenhance cell adhesion in a variety of other contexts. Within each of themethods described herein, one or more modulating agents may generally beadministered alone, or within a pharmaceutical composition. In eachspecific method described herein, as noted above, a targeting agent maybe employed to increase the local concentration of modulating agent atthe target site.

In general, within methods for modulating cell adhesion, acadherin-expressing cell is contacted with a modulating agent underconditions and for a time sufficient to permit inhibition or enhancementof a cadherin-mediated function. Cadherin-expressing cells include, butare not limited to, epithelial cells, endothelial cells, neural cells,tumor cells and lymphocytes. Such contact may be achieved in vitro, orin vivo by administration of a pharmaceutical composition as providedherein.

Within certain aspects, methods are provided in which cell adhesion isdiminished. In one such aspect, the present invention provides methodsfor reducing unwanted cellular adhesion by administering a modulatingagent as described herein. Unwanted cellular adhesion can occur betweentumor cells, between tumor cells and normal cells or between normalcells as a result of surgery, injury, chemotherapy, disease,inflammation or other condition jeopardizing cell viability or function.Preferred modulating agents for use within such methods include thosecomprising one or more of the sequences INPISGQ (SEQ ID NO:22), LNPISGQ(SEQ ID NO:23), IDPVSGQ (SEQ ID NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ(SEQ ID NO:26), KIDPVN (SEQ ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQID NO:52), PVSGR (SEQ ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ IDNO:55), IDPVN (SEQ ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ IDNO:58), CPISGQC (SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ IDNO:61), CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63) or CKIDPVC (SEQID NO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQ ID NO:87) in whichcyclization is indicated by an underline. Modulating agents mayalternatively, or in addition, comprise a derivative of one of theforegoing sequences. In addition, a modulating agent may comprise thesequence RGD, which is bound by integrins, the sequence LYHY (SEQ IDNO:70), which is bound by occludin, a JAM CAR sequence, a claudin CARsequence and/or one or more of HAV and/or a non-classical cadherin CARsequence. Preferably, such sequences are separated from the HAV-BMsequence via a linker. Alternatively, a separate modulator of celladhesion (e.g., integrin- and/or occludin-mediated) may be administeredin conjunction with the modulating agent(s), either within the samepharmaceutical composition or separately. Topical administration of themodulating agent(s) is generally preferred, but other means may also beemployed. Preferably, a fluid composition for topical administration(comprising, for example, physiological saline) comprises an amount ofmodulating agent as described above, and more preferably from 10 μg/mLto 1 mg/mL. Creams may generally be formulated as described above.Topical administration in the surgical field may be given once at theend of surgery by irrigation of the wound or as an intermittent orcontinuous irrigation with the use of surgical drains in thepost-operative period or by the use of drains specifically inserted inan area of inflammation, injury or disease in cases where surgery doesnot need to be performed. Alternatively, parenteral or transcutaneousadministration may be used to achieve similar results.

Within another such aspect, methods are provided for enhancing thedelivery of a drug through the skin of a mammal. Transdermal delivery ofdrugs is a convenient and non-invasive method that can be used tomaintain relatively constant blood levels of a drug. In general, tofacilitate drug delivery via the skin, it is necessary to perturbadhesion between the epithelial cells (keratinocytes) and theendothelial cells of the microvasculature. Using currently availabletechniques, only small, uncharged molecules may be delivered across skinin vivo. The methods described herein are not subject to the same degreeof limitation. Accordingly, a wide variety of drugs may be transportedacross the epithelial and endothelial cell layers of skin, for systemicor topical administration. Such drugs may be delivered to melanomas ormay enter the blood stream of the mammal for delivery to other siteswithin the body.

To enhance the delivery of a drug through the skin, a modulating agentas described herein and a drug are contacted with the skin surface.Preferred modulating agents for use within such methods include thosecomprising one or more of the sequences INPISGQ (SEQ ID NO:22), LNPISGQ(SEQ ID NO:23), IDPVSGQ (SEQ ID NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ(SEQ ID NO:26), KIDPVN (SEQ ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQID NO:52), PVSGR (SEQ ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ IDNO:55), IDPVN (SEQ ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ IDNO:58), CPISGQC (SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ IDNO:61), CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ IDNO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQ ID NO:87) in whichcyclization is indicated by an underline. Modulating agents mayalternatively, or in addition, comprise a derivative of one of theforegoing sequences. Multifunctional modulating agents comprising thecadherin CAR sequence HAV-BM linked to one or more of the Dsc or Dsg CARsequences may also be used to disrupt epithelial cell adhesion. Suchmodulating agents may also, or alternatively, comprise the fibronectinCAR sequence RGD, which is recognized by integrins, a JAM CAR sequence,a claudin CAR sequence and/or the occludin CAR sequence LYHY (SEQ IDNO:70). Alternatively, a separate modulator of cell adhesion may beadministered in conjunction with the modulating agent(s), either withinthe same pharmaceutical composition or separately.

Contact may be achieved by direct application of the modulating agent,generally within a composition formulated as a cream or gel, or usingany of a variety of skin contact devices for transdermal application(such as those described in European Patent Application No. 566,816 A;U.S. Pat. No. 5,613,958; U.S. Pat. No. 5,505,956). A skin patch providesa convenient method of administration (particularly for slow-releaseformulations). Such patches may contain a reservoir of modulating agentand drug separated from the skin by a membrane through which the drugdiffuses. Within other patch designs, the modulating agent and drug maybe dissolved or suspended in a polymer or adhesive matrix that is thenplaced in direct contact with the patient's skin. The modulating agentand drug may then diffuse from the matrix into the skin. Modulatingagent(s) and drug(s) may be contained within the same composition orskin patch, or may be separately administered, although administrationat the same time and site is preferred. In general, the amount ofmodulating agent administered via the skin varies with the nature of thecondition to be treated or prevented, but may vary as described above.Such levels may be achieved by appropriate adjustments to the deviceused, or by applying a cream formulated as described above. Transfer ofthe drug across the skin and to the target tissue may be predicted basedon in vitro studies using, for example, a Franz cell apparatus, andevaluated in vivo by appropriate means that will be apparent to those ofordinary skill in the art. As an example, monitoring of the serum levelof the administered drug over time provides an easy measure of the drugtransfer across the skin.

Transdermal drug delivery as described herein is particularly useful insituations in which a constant rate of drug delivery is desired, toavoid fluctuating blood levels of a drug. For example, morphine is ananalgesic commonly used immediately following surgery. When givenintermittently in a parenteral form (intramuscular, intravenous), thepatient usually feels sleepy during the first hour, is well during thenext 2 hours and is in pain during the last hour because the blood levelgoes up quickly after the injection and goes down below the desirablelevel before the 4 hour interval prescribed for re-injection is reached.Transdermal administration as described herein permits the maintenanceof constant levels for long periods of time (e.g., days), which allowsadequate pain control and mental alertness at the same time. Insulinprovides another such example. Many diabetic patients need to maintain aconstant baseline level of insulin which is different from their needsat the time of meals. The baseline level may be maintained usingtransdermal administration of insulin, as described herein. Antibioticsmay also be administered at a constant rate, maintaining adequatebactericidal blood levels, while avoiding the high levels that are oftenresponsible for the toxicity (e.g., levels of gentamycin that are toohigh typically result in renal toxicity).

Drug delivery by the methods of the present invention also provide amore convenient method of drug administration. For example, it is oftenparticularly difficult to administer parenteral drugs to newborns andinfants because of the difficulty associated with finding veins ofacceptable caliber to catheterize. However, newborns and infants oftenhave a relatively large skin surface as compared to adults. Transdermaldrug delivery permits easier management of such patients and allowscertain types of care that can presently be given only in hospitals tobe given at home. Other patients who typically have similar difficultieswith venous catheterization are patients undergoing chemotherapy orpatients on dialysis. In addition, for patients undergoing prolongedtherapy, transdermal administration as described herein is moreconvenient than parenteral administration.

Transdermal administration as described herein also allows thegastrointestinal tract to be bypassed in situations where parenteraluses would not be practical. For example, there is a growing need formethods suitable for administration of therapeutic small peptides andproteins, which are typically digested within the gastrointestinaltract. The methods described herein permit administration of suchcompounds and allow easy administration over long periods of time.Patients who have problems with absorption through theirgastrointestinal tract because of prolonged ileus or specificgastrointestinal diseases limiting drug absorption may also benefit fromdrugs formulated for transdermal application as described herein.

Further, there are many clinical situations where it is difficult tomaintain compliance. For example, patients with mental problems (e.g.,patients with Alzheimer's disease or psychosis) are easier to manage ifa constant delivery rate of drug is provided without having to rely ontheir ability to take their medication at specific times of the day.Also patients who simply forget to take their drugs as prescribed areless likely to do so if they merely have to put on a skin patchperiodically (e.g., every 3 days). Patients with diseases that arewithout symptoms, like patients with hypertension, are especially atrisk of forgetting to take their medication as prescribed.

For patients taking multiple drugs, devices for transdermal applicationsuch as skin patches may be formulated with combinations of drugs thatare frequently used together. For example, many heart failure patientsare given digoxin in combination with furosemide. The combination ofboth drugs into a single skin patch facilitates administration, reducesthe risk of errors (taking the correct pills at the appropriate time isoften confusing to older people), reduces the psychological strain oftaking “so many pills,” reduces skipped dosage because of irregularactivities and improves compliance.

The methods described herein are particularly applicable to humans, butalso have a variety of veterinary uses, such as the administration ofgrowth factors or hormones (e.g., for fertility control) to an animal.

As noted above, a wide variety of drugs may be administered according tothe methods provided herein. Some examples of drug categories that maybe administered transdermally include anti-inflammatory drugs (e.g., inarthritis and in other condition) such as all NSAID, indomethacin,prednisone, etc.; analgesics (especially when oral absorption is notpossible, such as after surgery, and when parenteral administration isnot convenient or desirable), including morphine, codeine, Demerol,acetaminophen and combinations of these (e.g., codeine plusacetaminophen); antibiotics such as Vancomycin (which is not absorbed bythe GI tract and is frequently given intravenously) or a combination ofINH and Rifampicin (e.g., for tuberculosis); anticoagulants such asheparin (which is not well absorbed by the GI tract and is generallygiven parenterally, resulting in fluctuation in the blood levels with anincreased risk of bleeding at high levels and risks of inefficacy atlower levels) and Warfarin (which is absorbed by the GI tract but cannotbe administered immediately after abdominal surgery because of thenormal ileus following the procedure); antidepressants (e.g., insituations where compliance is an issue as in Alzheimer's disease orwhen maintaining stable blood levels results in a significant reductionof anti-cholinergic side effects and better tolerance by patients), suchas amitriptylin, imipramin, prozac, etc.; antihypertensive drugs (e.g.,to improve compliance and reduce side effects associated withfluctuating blood levels), such as diuretics and beta-blockers (whichcan be administered by the same patch; e.g., furosemide and propanolol);antipsychotics (e.g., to facilitate compliance and make it easier forcare giver and family members to make sure that the drug is received),such as haloperidol and chlorpromazine; and anxiolytics or sedatives(e.g. to avoid the reduction of alertness related to high blood levelsafter oral administration and allow a continual benefit throughout theday by maintaining therapeutic levels constant).

Numerous other drugs may be administered as described herein, includingnaturally occurring and synthetic hormones, growth factors, proteins andpeptides. For example, insulin and human growth hormone, growth factorslike erythropoietin, interleukins and inteferons may be delivered viathe skin.

Kits for administering a drug via the skin of a mammal are also providedwithin the present invention. Such kits generally comprise a device fortransdermal application (e.g., a skin patch) in combination with, orimpregnated with, one or more modulating agents. A drug may additionallybe included within such kits.

Within a related aspect, the use of modulating agents as describedherein to increase the permeability of endothelial and epithelial celllayers, thereby facilitating sampling of the blood compartment bypassive diffusion. Such methods permit the detection and/or measurementof the levels of specific molecules circulating in the blood. Ingeneral, to sample the blood compartment, it is necessary to perturbadhesion between the epithelial cells (keratinocytes) and theendothelial cells of the microvasculature. Using currently availabletechniques, only small, uncharged molecules may be detected across skinin vivo. The methods described herein are not subject to the same degreeof limitation. Accordingly, a wide variety of blood components may besampled across epithelial and endothelial cell layers. Such sampling maybe achieved across any such cell layers, including skin and gums.

For example, application of one or more modulating agents to the skin,via a skin patch as described herein, permits the patch to function likea sponge to accumulate a small quantity of fluid containing arepresentative sample of the serum. The patch is then removed after aspecified amount of time and analyzed by suitable techniques for thecompound of interest (e.g., a medication, hormone, growth factor,metabolite or marker). Alternatively, a patch may be impregnated withreagents to permit a color change if a specific substance (e.g. anenzyme) is detected. Substances that can be detected in this mannerinclude, but are not limited to, illegal drugs such as cocaine, HIVenzymes, glucose and PSA. This technology is of particular benefit forhome testing kits.

To facilitate sampling of blood in a patient, a modulating agent asdescribed herein is contacted with the skin surface. Multifunctionalmodulating agents comprising an HAV-BM sequence linked to one or more ofthe OB-cadherin CAR sequence DDK, a claudin CAR sequence, the Dsc and/orDsg CAR sequences may also be used to disrupt epithelial cell adhesion.Such modulating agents may also, or alternatively, comprise thefibronectin CAR sequence RGD, which is recognized by integrins, aclaudin CAR sequence, a JAM CAR sequence and/or the occludin CARsequence LYHY (SEQ ID NO:70). Alternatively, a separate modulator ofnon-classical cadherin-mediated cell adhesion may be administered inconjunction with the modulating agent(s), either within the samepharmaceutical composition or separately.

Contact may be achieved as described herein for transdermal drugdelivery. Modulating agent(s) and reagents for assaying blood componentsmay, but need not, be contained within the same composition or skinpatch. In general, the amount of modulating agent administered via theskin may vary as described above. Such levels may be achieved byappropriate adjustments to the device used, or by applying a creamformulated as described above. Transfer of the blood component acrossthe skin may be predicted based on in vitro studies using, for example,a Franz cell apparatus, and evaluated in vivo by appropriate means thatwill be apparent to those of ordinary skill in the art.

Kits for sampling blood component via, for example, the skin or gums ofa mammal, are also provided within the present invention. Such kitsgenerally comprise a device for transdermal application (i.e., skinpatch) in combination with, or impregnated with, one or more modulatingagents. A reagent for detection of a blood component may additionally beincluded within such kits.

Within a further aspect, methods are provided for enhancing delivery ofa drug to a tumor in a mammal, comprising administering a modulatingagent in combination with a drug to a tumor-bearing mammal. Modulatingagents for use within such methods include those designed to disruptE-cadherin and/or N-cadherin mediated cell adhesion, such as thosecomprising one or more of the sequences INPISGQ (SEQ ID NO:22), LNPISGQ(SEQ ID NO:23), IDPVSGQ (SEQ ID NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ(SEQ ID NO:26), KIDPVN (SEQ ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQID NO:52), PVSGR (SEQ ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ IDNO:55), IDPVN (SEQ ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ IDNO:58), CPISGQC (SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ IDNO:61), CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ IDNO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQ ID NO:87) in whichcyclization is indicated by an underline. Modulating agents mayalternatively, or in addition, comprise a derivative of one of theforegoing sequences. Bi-functional modulating agents that comprise anHAV-BM sequence with flanking E-cadherin-specific sequences joined via alinker to an HAV-BM sequence with flanking N-cadherin-specific sequencesare also preferred. Preferably, the peptide portion(s) of a modulatingagent comprises 6-16 amino acids, since longer peptides are difficult todissolve in aqueous solution and are more likely to be degraded bypeptidases.

In one particularly preferred embodiment, a modulating agent is capableof disrupting cell adhesion mediated by multiple adhesion molecules. Forexample, a single branched modulating agent (or multiple agents linkedto a single molecule or support material) may disrupt E-cadherin,N-cadherin, occludin, Dsc and Dsg mediated cell adhesion, therebydisrupting adherens junctions, tight junctions and desmosomes. Such anagent may comprise one or more of the HAV-BM sequence, as well as a Dsgor Dsc CAR sequence; a JAM CAR sequence; a claudin CAR sequence; theOB-cadherin CAR sequence DDK; and the occludin CAR sequence LYHY (SEQ IDNO:70). Such agents serve as multifunctional disrupters of celladhesion. Alternatively, a separate modulator of non-classicalcadherin-mediated cell adhesion may be administered in conjunction withthe modulating agent(s), either within the same pharmaceuticalcomposition or separately. Preferred antibody modulating agents includeFab fragments directed against either the N-cadherin HAV-BM sequence orE-cadherin HAV-BM sequence. Fab fragments directed against the occludinCAR sequence GVNPTAQSSGSLYGSQIYALCNQFYTPAATGLYVDQYLYHYCVVDPQE (SEQ IDNO:69) may also be employed, either incorporated into a modulating agentor within a separate modulator that is administered concurrently.

Preferably, the modulating agent and the drug are formulated within thesame composition or drug delivery device prior to administration. Ingeneral, a modulating agent may enhance drug delivery to any tumor, andthe method of administration may be chosen based on the type of targettumor. For example, injection or topical administration as describedabove may be preferred for melanomas and other accessible tumors (e.g.,metastases from primary ovarian tumors may be treated by flushing theperitoneal cavity with the composition). Other tumors (e.g., bladdertumors) may be treated by injection of the modulating agent and the drug(such as mitomycin C) into the site of the tumor. In other instances,the composition may be administered systemically, and targeted to thetumor using any of a variety of specific targeting agents. Suitabledrugs may be identified by those of ordinary skill in the art based uponthe type of cancer to be treated (e.g., mitomycin C for bladder cancer).In general, the amount of modulating agent administered varies with themethod of administration and the nature of the tumor, within the typicalranges provided above, preferably ranging from about 1 μg/mL to about 2mg/mL, and more preferably from about 10 μg/mL to 1 mg/mL. Transfer ofthe drug to the target tumor may be evaluated by appropriate means thatwill be apparent to those of ordinary skill in the art. Drugs may alsobe labeled (e.g., using radionuclides) to permit direct observation oftransfer to the target tumor using standard imaging techniques.

Within a related aspect, the present invention provides methods fortreating cancer and/or inhibiting metastasis in a mammal. Cancer tumorsare solid masses of cells, growing out of control, which requirenourishment via blood vessels. The formation of new capillaries is aprerequisite for tumor growth and the emergence of metastases.Administration of modulating agents as described herein may disrupt thegrowth of such blood vessels, thereby providing effective therapy forthe cancer and/or inhibiting metastasis. Modulating agents may also beused to treat leukemias. Preferred modulating agents for use within suchmethods include those that disrupt N-cadherin and/or E-cadherin mediatedcell adhesion, such as those comprising one or more of the sequencesINPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ (SEQ ID NO:24),KIDPVNGQ (SEQ ID NO:25), PISGQ (SEQ ID NO:26), KIDPVN (SEQ ID NO:50),PVNGQ (SEQ ID NO:51), PISGQ (SEQ ID NO:52), PVSGR (SEQ ID NO:53), KIDPV(SEQ ID NO:54), KIDPVN (SEQ ID NO:55), IDPVN (SEQ ID NO:56), INPIS (SEQID NO:57), CPVNGQC (SEQ ID NO:58), CPISGQC (SEQ ID NO:59), CPVSGRC (SEQID NO:60), CKIDPVNC (SEQ ID NO:61), CIDPVNC (SEQ ID NO:62), CINPISC (SEQID NO:63), CKIDPVC (SEQ ID NO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQID NO:87), in which cyclization is indicated by an underline. Modulatingagents may alternatively, or in addition, comprise a derivative of oneof the foregoing sequences. Preferably, the peptide portion(s) of suchmodulating agents comprise 6-16 amino acids. In addition, a modulatingagent may comprise the sequence RGD, which is recognized by integrins, aJAM CAR sequence, a claudin CAR sequence, the occludin CAR sequence LYHY(SEQ ID NO:70), the OB-cadherin CAR sequence DDK, Dsc or Dsg CARsequences, and/or the occludin CAR sequence LYHY (SEQ ID NO:70).Preferably such sequences are separated from the HAV-BM sequence via alinker. Alternatively, a separate modulator of integrin- and/oroccludin-mediated cell adhesion may be administered in conjunction withthe modulating agents(s), either within the same pharmaceuticalcomposition or separately.

A modulating agent may be administered alone (e.g., via the skin) orwithin a pharmaceutical composition. For melanomas and certain otheraccessible tumors, injection or topical administration as describedabove may be preferred. For ovarian cancers, flushing the peritonealcavity with a composition comprising one or more modulating agents mayprevent metastasis of ovarian tumor cells. Other tumors (e.g., bladdertumors, bronchial tumors or tracheal tumors) may be treated by injectionof the modulating agent into the cavity. In other instances, thecomposition may be administered systemically, and targeted to the tumorusing any of a variety of specific targeting agents, as described above.In general, the amount of modulating agent administered varies dependingupon the method of administration and the nature of the cancer, but mayvary within the ranges identified above. The effectiveness of the cancertreatment or inhibition of metastasis may be evaluated using well knownclinical observations, such as the level of serum tumor markers (e.g.,CEA or PSA).

In yet another related aspect, the present invention provides methodsfor inducing apoptosis in a cadherin-expressing cell. In general,patients afflicted with cancer may benefit from such treatment. Certainpreferred modulating agents for use within such methods comprise one ormore of the sequences INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23),IDPVSGQ (SEQ ID NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ (SEQ ID NO:26),KIDPVN (SEQ ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQ ID NO:52), PVSGR(SEQ ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ ID NO:55), IDPVN (SEQID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ ID NO:58), CPISGQC (SEQ IDNO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ ID NO:61), CIDPVNC (SEQ IDNO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ ID NO:85), CINPC (SEQ IDNO:86) or CINPIC (SEQ ID NO:87), in which cyclization is indicated by anunderline. Modulating agents may alternatively, or in addition, comprisea derivative of one of the foregoing sequences. Modulating agentscomprising an additional CAR sequence (e.g., HAV, RGD, a Dsc CARsequence, a Dsg CAR sequence, a JAM CAR sequence, a claudin CAR sequenceand/or LYHY (SEQ ID NO:70)) are also preferred. As noted above, suchadditional sequences may be separated from the HAV-BM sequence via alinker. Alternatively, a separate modulator of integrin-mediated celladhesion may be administered in conjunction with the modulatingagent(s), either within the same pharmaceutical composition orseparately. Administration may be topical, via injection or by othermeans, and the addition of a targeting agent may be beneficial,particularly when the administration is systemic. Suitable modes ofadministration and dosages depend upon the location and nature of thecells for which induction of apoptosis is desired but, in general,dosages may vary as described above. A biopsy may be performed toevaluate the level of induction of apoptosis.

Within a further related aspect, a modulating agent may be used toinhibit angiogenesis (i.e., the growth of blood vessels frompre-existing blood vessels) in a mammal. Inhibition of angiogenesis maybe beneficial, for example, in patients afflicted with diseases such ascancer or arthritis. Preferred modulating agents for inhibition ofangiogenesis include those comprising one or more of the sequencesINPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ (SEQ ID NO:24),KIDPVNGQ (SEQ ID NO:25), PISGQ (SEQ ID NO:26), KIDPVN (SEQ ID NO:50),PVNGQ (SEQ ID NO:51), PISGQ (SEQ ID NO:52), PVSGR (SEQ ID NO:53), KIDPV(SEQ ID NO:54), KIDPVN (SEQ ID NO:55), IDPVN (SEQ ID NO:56), INPIS (SEQID NO:57), CPVNGQC (SEQ ID NO:58), CPISGQC (SEQ ID NO:59), CPVSGRC (SEQID NO:60), CKIDPVNC (SEQ ID NO:61), CIDPVNC (SEQ ID NO:62), CINPISC (SEQID NO:63), CKIDPVC (SEQ ID NO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQID NO:87), in which cyclization is indicated by an underline. Modulatingagents may alternatively, or in addition, comprise a derivative of oneof the foregoing sequences. In addition, a modulating agent for use ininhibiting angiogenesis may comprise the sequence RGD, which isrecognized by integrins, the OB-cadherin CAR sequence DDK, a JAM CARsequence, a claudin CAR sequence and/or the occludin CAR sequence LYHY(SEQ ID NO:70), separated from the HAV-BM sequence via a linker.Alternatively, a separate modulator of integrin- or occludin-mediatedcell adhesion may be administered in conjunction with the modulatingagent(s), either within the same pharmaceutical composition orseparately.

The effect of a particular modulating agent on angiogenesis maygenerally be determined by evaluating the effect of the agent on bloodvessel formation. Such a determination may generally be performed, forexample, using a chick chorioallantoic membrane assay (Iruela-Arispe etal., Molecular Biology of the Cell 6:327-343, 1995). Briefly, amodulating agent may be embedded in a mesh composed of vitrogen at oneor more concentrations (e.g., ranging from about 1 to 100 μg/mesh). Themesh(es) may then be applied to chick chorioallantoic membranes. After24 hours, the effect of the modulating agent may be determined usingcomputer assisted morphometric analysis. A modulating agent shouldinhibit angiogenesis by at least 25% at a concentration of 33 μg/mesh.

The addition of a targeting agent as described above may be beneficial,particularly when the administration is systemic. Suitable modes ofadministration and dosages depend upon the condition to be prevented ortreated but, in general, administration by injection is appropriate.Dosages may vary as described above. The effectiveness of the inhibitionmay be evaluated grossly by assessing the inability of the tumors tomaintain their growth and microscopically by observing an absence ofnerves at the periphery of the tumor.

The present invention also provides methods for enhancing drug deliveryto the central nervous system of a mammal. The blood/brain barrier islargely impermeable to most neuroactive agents, and delivery of drugs tothe brain of a mammal often requires invasive procedures. Using amodulating agent as described herein, however, delivery may be by, forexample, systemic administration of a modulating agent-drug-targetingagent combination, injection of a modulating agent (alone or incombination with a drug and/or targeting agent) into the carotid arteryor application of a skin patch comprising a modulating agent to the headof the patient. Certain preferred modulating agents for use within suchmethods comprise one or more of the sequences INPISGQ (SEQ ID NO:22),LNPISGQ (SEQ ID NO:23), IDPVSGQ (SEQ ID NO:24), KIDPVNGQ (SEQ ID NO:25),PISGQ (SEQ ID NO:26), KIDPVN (SEQ ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ(SEQ ID NO:52), PVSGR (SEQ ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQID NO:55), IDPVN (SEQ ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ IDNO:58), CPISGQC (SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ IDNO:61), CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ IDNO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQ ID NO:87), in whichcyclization is indicated by an underline. Modulating agents mayalternatively, or in addition, comprise a derivative of one of theforegoing sequences. Also preferred are multi-functional modulatingagents comprising an HAV-BM sequence and the occludin CAR sequence LYHY(SEQ ID NO:70), a JAM CAR sequence and/or a claudin CAR sequence,preferably joined by a linker. Alternatively, a separate modulator ofoccludin-mediated cell adhesion may be administered in conjunction withthe modulating agent(s), either within the same pharmaceuticalcomposition or separately. Preferably, the peptide portion(s) of suchmodulating agents comprise 6-16 amino acids. Modulating agents mayfurther comprise antibodies or Fab fragments directed against anN-cadherin HAV-BM sequence. Fab fragments directed against the occludinCAR sequence GVNPTAQSSGSLYGSQIYALCNQFYTPAATGLYVDQYLYHYCVVDPQE (SEQ IDNO:69) may also be employed, either incorporated into the modulatingagent or administered concurrently as a separate modulator.

In general, the amount of modulating agent administered varies with themethod of administration and the nature of the condition to be treatedor prevented, but typically varies as described above. Transfer of thedrug to the central nervous system may be evaluated by appropriate meansthat will be apparent to those of ordinary skill in the art, such asmagnetic resonance imaging (MRI) or PET scan (positron emittedtomography).

In certain other aspects, the present invention provides methods forenhancing adhesion of cadherin-expressing cells. Within certainembodiments, a modulating agent may be linked to a solid support,resulting in a matrix that comprises multiple modulating agents. Withinone such embodiment, the support is a polymeric matrix to whichmodulating agents and molecules comprising other CAR sequence(s) areattached (e.g. modulating agents and molecules comprising an RGDsequence may be attached to the same matrix, preferably in analternating pattern). Such matrices may be used in contexts in which itis desirable to enhance adhesion mediated by multiple cell adhesionmolecules. Alternatively, the modulating agent itself may comprisemultiple HAV-BM sequences or antibodies (or fragments thereof),separated by linkers as described above. Either way, the modulatingagent(s) function as a “biological glue” to bind multiplecadherin-expressing cells within a variety of contexts.

Within one aspect, such modulating agents may be used to enhance woundhealing and/or reduce scar tissue in a mammal. Peptides that may belinked to a support, and/or to one another via a linker, to generate asuitable modulating agent include, but are not limited to, thosecomprising one or more of the sequences INPISGQ (SEQ ID NO:22), LNPISGQ(SEQ ID NO:23), IDPVSGQ (SEQ ID NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ(SEQ ID NO:26), KIDPVN (SEQ ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQID NO:52), PVSGR (SEQ ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ IDNO:55), IDPVN (SEQ ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ IDNO:58), CPISGQC (SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ IDNO:61), CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ IDNO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQ ID NO:87), in whichcyclization is indicated by an underline. Modulating agents mayalternatively, or in addition, comprise a derivative of one of theforegoing sequences. Modulating agents that are linked to abiocompatible and biodegradable matrix such as cellulose or collagen areparticularly preferred. For use within such methods, a modulating agentshould have a free amino or hydroxyl group. Multi-functional modulatingagents comprising the HAV-BM sequence, the fibronectin CAR sequence RGD,which is recognized by integrins, the OB-cadherin CAR sequence DDK, aJAM CAR sequence and/or a Dsc or Dsg CAR sequence may also be used aspotent stimulators of wound healing and/or to reduce scar tissue. Suchagents may also, or alternatively, comprise the occludin CAR sequenceLYHY (SEQ ID NO:70) and/or a claudin CAR sequence. Alternatively, one ormore separate modulator of integrin-, Dsc-, Dsg- and/oroccludin-mediated cell adhesion may be administered in conjunction withthe modulating agent(s), either within the same pharmaceuticalcomposition or separately.

The modulating agents are generally administered topically to the wound,where they may facilitate closure of the wound and may augment, or evenreplace, stitches. Similarly, administration of matrix-linked modulatingagents may facilitate cell adhesion in foreign tissue implants (e.g.,skin grafting and prosthetic implants) and may prolong the duration andusefulness of collagen injection. In general, the amount ofmatrix-linked modulating agent administered to a wound, graft or implantsite varies with the severity of the wound and/or the nature of thewound, graft, or implant, but may vary as discussed above.

Within another aspect, one or more modulating agents may be linked tothe interior surface of a tissue culture plate or other cell culturesupport, such as for use in a bioreactor. Such linkage may be performedby any suitable technique, as described above. Modulating agents linkedin this fashion may generally be used to immobilize cadherin-expressingcells. For example, dishes or plates coated with one or more modulatingagents may be used to immobilize cadherin-expressing cells within avariety of assays and screens. Within bioreactors (i.e., systems forlarge scale production of cells or organoids), modulating agents maygenerally be used to improve cell attachment and stabilize cell growth.Modulating agents may also be used within bioreactors to support theformation and function of highly differentiated organoids derived, forexample, from dispersed populations of fetal mammalian cells.Bioreactors containing biomatrices of modulating agent(s) may also beused to facilitate the production of specific proteins.

Modulating agents as described herein may be used within a variety ofbioreactor configurations. In general, a bioreactor is designed with aninterior surface area sufficient to support large numbers of adherentcells. This surface area can be provided using membranes, tubes,microtiter wells, columns, hollow fibers, roller bottles, plates,dishes, beads or a combination thereof. A bioreactor may becompartmentalized. The support material within a bioreactor may be anysuitable material known in the art; preferably, the support materialdoes not dissolve or swell in water. Preferred support materialsinclude, but are not limited to, synthetic polymers such as acrylics,vinyls, polyethylene, polypropylene, polytetrafluoroethylene, nylons,polyurethanes, polyamides, polysulfones and poly(ethyleneterephthalate); ceramics; glass and silica.

The present invention also provides, within further aspects, methods forenhancing and/or directing neurological growth. In one such aspect,neurite outgrowth may be enhanced and/or directed by contacting a neuronwith one or more modulating agents. Preferred modulating agents for usewithin such methods are linked to a polymeric matrix or other supportand/or contain multiple HAV-BM sequences separated by one or morelinkers. Peptides that may be linked to a support material (and/or toone another via a linker to generate a suitable modulating agent)include, but are not limited to, those comprising one or more of thesequences INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ (SEQID NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ (SEQ ID NO:26), KIDPVN (SEQ IDNO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQ ID NO:52), PVSGR (SEQ IDNO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ ID NO:55), IDPVN (SEQ IDNO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ ID NO:58), CPISGQC (SEQ IDNO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ ID NO:61), CIDPVNC (SEQ IDNO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ ID NO:85), CINPC (SEQ IDNO:86) or CINPIC (SEQ ID NO:87), in which cyclization is indicated by anunderline. Certain preferred modulating agents comprise one or more ofN-Ac-INPISGQ-NH₂ (SEQ ID NO:22), H—INPISGQ-OH (SEQ ID NO:22),N-Ac-NLKIDPVNGQI-NH₂ (SEQ ID NO:20), H-WLKIDPVNGQI-OH (SEQ ID NO:13),H-LKIDPVNGQI-OH (SEQ ID NO:21), H-LKIDPANGQI-OH (SEQ ID NO:64) orH-LKIDAVNGQI-OH (SEQ ID NO:65).

Modulating agents may alternatively, or in addition, comprise aderivative of one of the foregoing sequences. In addition, a modulatingagent comprising HAV, RGD and/or YIGSR (SEQ ID NO:66), which are boundby integrins, and/or the N-CAM CAR sequence KYSFNYDGSE (SEQ ID NO:67)may further facilitate neurite outgrowth. Modulating agents comprisingantibodies, or fragments thereof, may be used within this aspect of thepresent invention without the use of linkers or support materials.Preferred antibody modulating agents include Fab fragments directedagainst an N-cadherin HAV-BM sequence. Fab fragments directed againstthe N-CAM CAR sequence KYSFNYDGSE (SEQ ID NO:67) may also be employed,either incorporated into the modulating agent or administeredconcurrently as a separate modulator.

The method of achieving contact and the amount of modulating agent usedwill depend upon the location of the neuron and the extent and nature ofthe outgrowth desired. For example, a neuron may be contacted (e.g., viaimplantation) with modulating agent(s) linked to a support material suchas a suture, fiber nerve guide or other prosthetic device such that theneurite outgrowth is directed along the support material. Alternatively,a tubular nerve guide may be employed, in which the lumen of the nerveguide contains a composition comprising the modulating agent(s). Invivo, such nerve guides or other supported modulating agents may beimplanted using well known techniques to, for example, facilitate thegrowth of severed neuronal connections and/or to treat spinal cordinjuries. It will be apparent to those of ordinary skill in the art thatthe structure and composition of the support should be appropriate forthe particular injury being treated. In vitro, a polymeric matrix maysimilarly be used to direct the growth of neurons onto patternedsurfaces as described, for example, in U.S. Pat. No. 5,510,628.

Within another aspect, one or more modulating agents may be used fortherapy of a demyelinating neurological disease in a mammal. There are anumber of demyelinating diseases, such as multiple sclerosis,characterized by oligodendrocyte death. Since Schwann cell migration onastrocytes is inhibited by N-cadherin, modulating agents that disruptN-cadherin mediated cell adhesion as described herein, when implantedwith Schwann cells into the central nervous system, may facilitateSchwann cell migration and permit the practice of Schwann cellreplacement therapy.

Multiple sclerosis patients suitable for treatment may be identified bycriteria that establish a diagnosis of clinically definite or clinicallyprobable MS (see Poser et al., Ann. Neurol. 13:227, 1983). Candidatepatients for preventive therapy may be identified by the presence ofgenetic factors, such as HLA-type DR2a and DR2b, or by the presence ofearly disease of the relapsing remitting type.

Schwann cell grafts may be implanted directly into the brain along withthe modulating agent(s) using standard techniques. Preferred peptidemodulating agents for use within such methods include those comprisingone or more of the sequences INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ IDNO:23), IDPVSGQ (SEQ ID NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ (SEQ IDNO:26), KIDPVN (SEQ ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQ IDNO:52), PVSGR (SEQ ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ IDNO:55), IDPVN (SEQ ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ IDNO:58), CPISGQC (SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ IDNO:61), CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ IDNO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQ ID NO:87), in whichcyclization is indicated by an underline. Modulating agents mayalternatively, or in addition, comprise a derivative of one of theforegoing sequences. Modulating agents may further comprise HAV, RGDand/or YIGSR (SEQ ID NO:66), which are bound by integrins, and/or theN-CAM CAR sequence KYSFNYDGSE (SEQ ID NO:67). Preferred antibodymodulating agents include Fab fragments directed against the N-cadherinCAR sequence KYSFNYDGSE (SEQ ID NO:67). Such antibodies and fragmentscan be prepared using standard techniques, as discussed above. Suitableamounts of modulating agent generally range as described above,preferably from about 10 μg/mL to about 1 mg/mL.

Alternatively, a modulating agent may be implanted with oligodendrocyteprogenitor cells (OPs) derived from donors not afflicted with thedemyelinating disease. The myelinating cell of the CNS is theoligodendrocyte. Although mature oligodendrocytes and immature cells ofthe oligodendrocyte lineage, such as the oligodendrocyte type 2astrocyte progenitor, have been used for transplantation, OPs are morewidely used. OPs are highly motile and are able to migrate fromtransplant sites to lesioned areas where they differentiate into maturemyelin-forming oligodendrocytes and contribute to repair of demyelinatedaxons (see e.g. Groves et al., Nature 362:453-55, 1993; Baron-VanEvercooren et al., Glia 16:147-64, 1996). OPs can be isolated usingroutine techniques known in the art (see e.g. Milner andFrench-Constant, Development 120:3497-3506, 1994), from many regions ofthe CNS including brain, cerebellum, spinal cord, optic nerve andolfactory bulb. Substantially greater yields of OP's are obtained fromembryonic or neonatal rather than adult tissue. OPs may be isolated fromhuman embryonic spinal cord and cultures of neurospheres established.Human fetal tissue is a potential valuable and renewable source of donorOP's for future, long range transplantation therapies of demyelinatingdiseases such as MS.

OPs can be expanded in vitro if cultured as “homotypic aggregates” or“spheres” (Avellana-Adalid et al., J. Neurosci. Res. 45:558-70, 1996).Spheres (sometimes called “oligospheres” or “neurospheres”) are formedwhen OPs are grown in suspension in the presence of growth factors suchas PDGF and FGF. OPs can be harvested from spheres by mechanicaldissociation and used for subsequent transplantation or establishment ofnew spheres in culture. Alternatively, the spheres themselves may betransplanted, providing a “focal reservoir” of OPs (Avellana-Adalid etal., J. Neurosci. Res. 45:558-70, 1996).

An alternative source of OP may be spheres derived from CNS stem cells.Recently, Reynolds and Weiss, Dev. Biol. 165:1-13, 1996 have describedspheres formed from EGF-responsive cells derived from embryonicneuroepithelium, which appear to retain the pluripotentiality exhibitedby neuroepithelium in vivo. Cells dissociated from these spheres areable to differentiate into neurons, oligodendrocytes and astrocytes whenplated on adhesive substrates in the absence of EGF, suggesting thatEGF-responsive cells derived from undifferentiated embryonicneuroepithelium may represent CNS stem cells (Reynolds and Weiss, Dev.Biol. 165:1-13, 1996). Spheres derived from CNS stem cells provide analternative source of OP which may be manipulated in vitro fortransplantation in vivo. Spheres composed of CNS stem cells may furtherprovide a microenvironment conducive to increased survival, migration,and differentiation of the OPs in vivo.

The use of neurospheres for the treatment of MS may be facilitated bymodulating agents that enhance cell migration from the spheres. In theabsence of modulating agent, the cells within the spheres adhere tightlyto one another and migration out of the spheres is hindered. Modulatingagents that disrupt N-cadherin mediated cell adhesion as describedherein, when injected with neurospheres into the central nervous system,may improve cell migration and increase the efficacy of OP replacementtherapy.

Neurosphere grafts may be implanted directly into the central nervoussystem along with the modulating agent(s) using standard techniques.Preferred peptide modulating agents for use within such methods includethose comprising one or more of the sequences INPISGQ (SEQ ID NO:22),LNPISGQ (SEQ ID NO:23), IDPVSGQ (SEQ ID NO:24), KIDPVNGQ (SEQ ID NO:25),PISGQ (SEQ ID NO:26), KIDPVN (SEQ ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ(SEQ ID NO:52), PVSGR (SEQ ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQID NO:55), IDPVN (SEQ ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ IDNO:58), CPISGQC (SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ IDNO:61), CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ IDNO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQ ID NO:87), in whichcyclization is indicated by an underline. Modulating agents mayalternatively, or in addition, comprise a derivative of one of theforegoing sequences. Modulating agents comprising one or more of thesesequences or derivatives thereof are also preferred. Preferred antibodymodulating agents include Fab fragments directed against an N-cadherinHAV-BM sequence (e.g., INPISGQ (SEQ ID NO:22)). Such antibodies andfragments can be prepared using standard techniques, as discussed above.Suitable amounts of modulating agent generally range as described above,preferably from about 10 μg/mL to about 1 mg/mL.

Alternatively, a modulating agent may be administered alone or within apharmaceutical composition. The duration and frequency of administrationwill be determined by such factors as the condition of the patient, andthe type and severity of the patient's disease. Within particularlypreferred embodiments of the invention, the modulating agent orpharmaceutical composition may be administered at a dosage ranging from0.1 mg/kg to 20 mg/kg although appropriate dosages may be determined byclinical trials. Methods of administration include injection,intravenous or intrathecal (i.e., directly in cerebrospinal fluid). Amodulating agent or pharmaceutical composition may further comprise adrug (e.g., an immunomodulatory drug).

Effective treatment of multiple sclerosis may be evidenced by any of thefollowing criteria: EDSS (extended disability status scale), appearanceof exacerbations or MRI (magnetic resonance imaging). The EDSS is ameans to grade clinical impairment due to MS (Kurtzke, Neurolog 33:1444,1983), and a decrease of one full step defines an effective treatment inthe context of the present invention (Kurtzke, Ann. Neurol. 36:573-79,1994). Exacerbations are defined as the appearance of a new symptom thatis attributable to MS and accompanied by an appropriate new neurologicabnormality (Sipe et al., Neurology 34:1368, 1984). Therapy is deemed tobe effective if there is a statistically significant difference in therate or proportion of exacerbation-free patients between the treatedgroup and the placebo group or a statistically significant difference inthe time to first exacerbation or duration and severity in the treatedgroup compared to control group. MRI can be used to measure activelesions using gadolinium-DTPA-enhanced imaging (McDonald et al. Ann.Neurol. 36:14, 1994) or the location and extent of lesions usingT₂-weighted techniques. The presence, location and extent of MS lesionsmay be determined by radiologists using standard techniques. Improvementdue to therapy is established when there is a statistically significantimprovement in an individual patient compared to baseline or in atreated group versus a placebo group.

Efficacy of the modulating agent in the context of prevention may bejudged based on clinical measurements such as the relapse rate and EDSS.Other criteria include a change in area and volume of T2 images on MRI,and the number and volume of lesions determined by gadolinium enhancedimages.

Within further aspects, modulating agents as described herein may beused for modulating the immune system of a mammal in any of severalways. Cadherins are expressed on immature B and T cells (thymocytes andbone marrow pre-B cells), as well as on specific subsets of activated Band T lymphocytes and some hematological malignancies (see Lee et al.,J. Immunol. 152:5653-5659, 1994; Munro et al., Cellular Immunol.169:309-312, 1996; Tsutsui et al., J. Biochem. 120:1034-1039, 1996;Cepek et al., Proc. Natl. Acad. Sci. USA 93:6567-6571, 1996). Modulatingagents may generally be used to modulate specific steps within cellularinteractions during an immune response or during the dissemination ofmalignant lymphocytes.

For example, a modulating agent as described herein may be used to treatdiseases associated with excessive generation of otherwise normal Tcells. Without wishing to be bound by any particular theory, it isbelieved that the interaction of cadherins on maturing T cells and Bcell subsets contributes to protection of these cells from programmedcell death. A modulating agent may decrease such interactions, leadingto the induction of programmed cell death. Accordingly, modulatingagents may be used to treat certain types of diabetes and rheumatoidarthritis, particularly in young children where the cadherin expressionon thymic pre-Tcells is greatest.

Modulating agents may also be administered to patients afflicted withcertain skin disorders (such as cutaneous lymphomas), acute B cellleukemia and excessive immune reactions involving the humoral immunesystem and generation of immunoglobulins, such as allergic responses andantibody-mediated graft rejection. In addition, patients withcirculating cadherin-positive malignant cells (e.g., during regimeswhere chemotherapy or radiation therapy is eliminating a major portionof the malignant cells in bone marrow and other lymphoid tissue) maybenefit from treatment with a modulating agent. Such treatment may alsobenefit patients undergoing transplantation with peripheral blood stemcells.

Preferred modulating agents for use within such methods include thosethat disrupt E-cadherin and/or N-cadherin mediated cell adhesion, suchas those comprising one or more of the sequences INPISGQ (SEQ ID NO:22),LNPISGQ (SEQ ID NO:23), IDPVSGQ (SEQ ID NO:24), KIDPVNGQ (SEQ ID NO:25),PISGQ (SEQ ID NO:26), KIDPVN (SEQ ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ(SEQ ID NO:52), PVSGR (SEQ ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQID NO:55), IDPVN (SEQ ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ IDNO:58), CPISGQC (SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ IDNO:61), CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ IDNO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQ ID NO:87), in whichcyclization is indicated by an underline. Modulating agents mayalternatively, or in addition, comprise a derivative of one of theforegoing sequences. In addition, a preferred modulating agent maycomprise one or more additional CAR sequences, such as HAV, RGD and/orKYSFNYDGSE (SEQ ID NO:67). As noted above, such additional sequence(s)may be separated from the HAV-BM sequence via a linker. Alternatively, aseparate modulator of integrin-mediated cell adhesion may beadministered in conjunction with the modulating agent(s), either withinthe same pharmaceutical composition or separately.

Within the above methods, the modulating agent(s) are preferablyadministered systemically (usually by injection) or topically. Amodulating agent may be linked to a targeting agent. For example,targeting to the bone marrow may be beneficial. A suitable dosage issufficient to effect a statistically significant reduction in thepopulation of B and/or T cells that express cadherin and/or animprovement in the clinical manifestation of the disease being treated.Typical dosages generally range as described above.

Within further aspects, the present invention provides methods and kitsfor preventing pregnancy in a mammal. In general, disruption ofE-cadherin function prevents the adhesion of trophoblasts and theirsubsequent fusion to form syncitiotrophoblasts. In one embodiment, oneor more modulating agents as described herein may be incorporated intoany of a variety of well known contraceptive devices, such as spongessuitable for intravaginal insertion (see, e.g., U.S. Pat. No. 5,417,224)or capsules for subdermal implantation. Other modes of administrationare possible, however, including transdermal administration, formodulating agents linked to an appropriate targeting agent. Preferredmodulating agents for use within such methods include those comprisingone or more of the sequences INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ IDNO:23), IDPVSGQ (SEQ ID NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ (SEQ IDNO:26), KIDPVN (SEQ ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQ IDNO:52), PVSGR (SEQ ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ IDNO:55), IDPVN (SEQ ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ IDNO:58), CPISGQC (SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ IDNO:61), CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ IDNO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQ ID NO:87), in whichcyclization is indicated by an underline. Modulating agents mayalternatively, or in addition, comprise a derivative of one of theforegoing sequences. In addition, a preferred modulating agent maycomprise additional CAR sequences, such as HAV, DDK and/or RGD. As notedabove, such additional sequences may be separated from the HAV-BMsequence via a linker. Alternatively, a separate modulator ofintegrin-mediated cell adhesion may be administered in conjunction withthe modulating agent(s), either within the same pharmaceuticalcomposition or separately.

Suitable methods for incorporation into a contraceptive device dependupon the type of device and are well known in the art. Such devicesfacilitate administration of the modulating agent(s) to the uterineregion and may provide a sustained release of the modulating agent(s).In general, modulating agent(s) may be administered via such acontraceptive device at a dosage ranging from 0.1 to 50 mg/kg, althoughappropriate dosages may be determined by monitoring hCG levels in theurine. hCG is produced by the placenta, and levels of this hormone risein the urine of pregnant women. The urine hCG levels can be assessed byradio-immunoassay using well known techniques. Kits for preventingpregnancy generally comprise a contraceptive device impregnated with oneor more modulating agents.

Alternatively, a sustained release formulation of one or more modulatingagents may be implanted, typically subdermally, in a mammal for theprevention of pregnancy. Such implantation may be performed using wellknown techniques. Preferably, the implanted formulation provides adosage as described above, although the minimum effective dosage may bedetermined by those of ordinary skill in the art using, for example, anevaluation of hCG levels in the urine of women.

The present invention also provides methods for increasingvasopermeability in a mammal by administering one or more modulatingagents or pharmaceutical compositions. Within blood vessels, endothelialcell adhesion (mediated by N-cadherin) results in decreased vascularpermeability. Accordingly, modulating agents as described herein thatdecrease N-cadherin mediated adhesion may be used to increase vascularpermeability. Particularly preferred modulating agents include thosecomprising one or more of the sequences INPISGQ (SEQ ID NO:22), LNPISGQ(SEQ ID NO:23), IDPVSGQ (SEQ ID NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ(SEQ ID NO:26), KIDPVN (SEQ ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQID NO:52), PVSGR (SEQ ID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ IDNO:55), IDPVN (SEQ ID NO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ IDNO:58), CPISGQC (SEQ ID NO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ IDNO:61), CIDPVNC (SEQ ID NO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ IDNO:85), CINPC (SEQ ID NO:86) or CINPIC (SEQ ID NO:87), in whichcyclization is indicated by an underline. Modulating agents mayalternatively, or in addition, comprise a derivative of one of theforegoing sequences. In addition, a preferred modulating agent maycomprise an occludin CAR sequence LYHY (SEQ ID NO:70), a JAM CARsequence, a claudin CAR sequence and/or an OB-cadherin CAR sequence DDK.As noted above, such an additional sequence may be separated from theHAV sequence via a linker. Alternatively, a separate modulator ofoccludin mediated cell adhesion may be administered in conjunction withone or modulating agents, either within the same pharmaceuticalcomposition or separately.

Within certain embodiments, preferred modulating agents for use withinsuch methods include peptides capable of decreasing both endothelial andtumor cell adhesion. Such modulating agents may be used to facilitatethe penetration of anti-tumor therapeutic or diagnostic agents (e.g.,monoclonal antibodies) through endothelial cell permeability barriersand tumor barriers. For example, a modulating agent may further comprisean E-cadherin HAV or HAV-BM sequence. Alternatively, separate modulatingagents capable of disrupting N- and E-cadherin mediated adhesion may beadministered concurrently.

In one particularly preferred embodiment, a modulating agent is furthercapable of disrupting cell adhesion mediated by multiple adhesionmolecules. Such an agent may comprise an HAV-BM sequence, as well as anRGD sequence, a Dsc CAR sequence, a Dsg CAR sequence and/or the occludinCAR sequence LYHY (SEQ ID NO:70). Alternatively, a separate modulatorcell adhesion that comprises a CAR sequence other than an HAV-BMsequence may be administered in conjunction with the modulatingagent(s), either within the same pharmaceutical composition orseparately.

Treatment with a modulating agent may be appropriate, for example, priorto administration of an anti-tumor therapeutic or diagnostic agent(e.g., a monoclonal antibody or other macromolecule), an antimicrobialagent or an anti-inflammatory agent, in order to increase theconcentration of such agents in the vicinity of the target tumor,organism or inflammation without increasing the overall dose to thepatient. Modulating agents for use within such methods may be linked toa targeting agent to further increase the local concentration ofmodulating agent, although systemic administration of a vasoactive agenteven in the absence of a targeting agent increases the perfusion ofcertain tumors relative to other tissues. Suitable targeting agentsinclude antibodies and other molecules that specifically bind to tumorcells or to components of structurally abnormal blood vessels. Forexample, a targeting agent may be an antibody that binds to a fibrindegradation product or a cell enzyme such as a peroxidase that isreleased by granulocytes or other cells in necrotic or inflamed tissues.

Administration via intravenous injection or transdermal administrationis generally preferred. Effective dosages are generally sufficient toincrease localization of a subsequently administered diagnostic ortherapeutic agent to an extent that improves the clinical efficacy oftherapy of accuracy of diagnosis to a statistically significant degree.Comparison may be made between treated and untreated tumor host animalsto whom equivalent doses of the diagnostic or therapeutic agent areadministered. In general, dosages range as described above.

Within a further aspect, modulating agents as described herein may beused for controlled inhibition of synaptic stability, resulting inincreased synaptic plasticity. Within this aspect, administration of oneor more modulating agents may be advantageous for repair processeswithin the brain, as well as learning and memory, in which neuralplasticity is a key early event in the remodeling of synapses. Celladhesion molecules, particularly N-cadherin and E-cadherin, can functionto stabilize synapses, and loss of this function is thought to be theinitial step in the remodeling of the synapse that is associated withlearning and memory (Doherty et al., J. Neurobiology, 26:437-446, 1995;Martin and Kandel, Neuron, 17:567-570, 1996; Fannon and Colman, Neuron,17:423-434, 1996). Inhibition of cadherin function by administration ofone or more modulating agents that inhibit cadherin function maystimulate learning and memory. Preferred modulating agents for usewithin such methods include those that disrupt E-cadherin and/orN-cadherin mediated cell adhesion, such as those comprising one or moreof the sequences INPISGQ (SEQ ID NO:22), LNPISGQ (SEQ ID NO:23), IDPVSGQ(SEQ ID NO:24), KIDPVNGQ (SEQ ID NO:25), PISGQ (SEQ ID NO:26), KIDPVN(SEQ ID NO:50), PVNGQ (SEQ ID NO:51), PISGQ (SEQ ID NO:52), PVSGR (SEQID NO:53), KIDPV (SEQ ID NO:54), KIDPVN (SEQ ID NO:55), IDPVN (SEQ IDNO:56), INPIS (SEQ ID NO:57), CPVNGQC (SEQ ID NO:58), CPISGQC (SEQ IDNO:59), CPVSGRC (SEQ ID NO:60), CKIDPVNC (SEQ ID NO:61), CIDPVNC (SEQ IDNO:62), CINPISC (SEQ ID NO:63), CKIDPVC (SEQ ID NO:85), CINPC (SEQ IDNO:86) or CINPIC (SEQ ID NO:87), in which cyclization is indicated by anunderline. Modulating agents may alternatively, or in addition, comprisea derivative of one of the foregoing sequences. In addition, a preferredmodulating agent may comprise one or more additional CAR sequences, suchas the sequence RGD, which is bound by integrins and/or the N-CAM CARsequence KYSFNYDGSE (SEQ ID NO:67). As noted above, such additionalsequence(s) may be separated from the HAV-BM sequence via a linker.Alternatively, a separate modulator of integrin and/or N-CAM mediatedcell adhesion may be administered in conjunction with the modulatingagent(s), either within the same pharmaceutical composition orseparately. For such aspects, administration may be via encapsulationinto a delivery vehicle such as a liposome, using standard techniques,and injection into, for example, the carotid artery. Alternatively, amodulating agent may be linked to a disrupter of the blood-brainbarrier. In general dosages range as described above.

Assays Employing Anti-HAV-BM Antibodies

Other aspects of the present invention provide methods that employantibodies raised against an HAV-BM sequence for diagnostic and assaypurposes. Such polyclonal and monoclonal antibodies may be raisedagainst a peptide using conventional techniques and as described above.Assays employing antibodies typically involve using an antibody todetect the presence or absence of a cadherin (free or on the surface ofa cell), or proteolytic fragment containing the EC1 or EC4 domain in asuitable biological sample, such as tumor or normal tissue biopsies,blood, lymph node, serum or urine samples, or other tissue, homogenate,or extract thereof obtained from a patient.

There are a variety of assay formats known to those of ordinary skill inthe art for using an antibody to detect a target molecule in a sample.See, e.g. Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988. For example, the assay may be performed in aWestern blot format, wherein a protein preparation from the biologicalsample is submitted to gel electrophoresis, transferred to a suitablemembrane and allowed to react with the antibody. The presence of theantibody on the membrane may then be detected using a suitable detectionreagent, as described below.

In another embodiment, the assay involves the use of antibodyimmobilized on a solid support to bind to the target cadherin, or aproteolytic fragment containing the EC1 or EC4 domain and encompassingthe CAR sequence, and remove it from the remainder of the sample. Thebound cadherin may then be detected using a second antibody or reagentthat contains a reporter group. Alternatively, a competitive assay maybe utilized, in which a cadherin is labeled with a reporter group andallowed to bind to the immobilized antibody after incubation of theantibody with the sample. The extent to which components of the sampleinhibit the binding of the labeled cadherin to the antibody isindicative of the reactivity of the sample with the immobilizedantibody, and as a result, indicative of the level of the cadherin inthe sample.

The solid support may be any material known to those of ordinary skillin the art to which the antibody may be attached, such as a test well ina microtiter plate, a nitrocellulose filter or another suitablemembrane. Alternatively, the support may be a bead or disc, such asglass, fiberglass, latex or a plastic such as polystyrene orpolyvinylchloride. The antibody may be immobilized on the solid supportusing a variety of techniques known to those in the art, which are amplydescribed in the patent and scientific literature.

In certain embodiments, the assay for detection of a cadherin in asample is a two-antibody sandwich assay. This assay may be performed byfirst contacting an antibody that has been immobilized on a solidsupport, commonly the well of a microtiter plate, with the biologicalsample, such that the cadherin within the sample is allowed to bind tothe immobilized antibody (a 30 minute incubation time at roomtemperature is generally sufficient). Unbound sample is then removedfrom the immobilized cadherin-antibody complexes and a second antibody(containing a reporter group such as an enzyme, dye, radionuclide,luminescent group, fluorescent group or biotin) capable of binding to adifferent site on the cadherin is added. The amount of second antibodythat remains bound to the solid support is then determined using amethod appropriate for the specific reporter group. The method employedfor detecting the reporter group depends upon the nature of the reportergroup. For radioactive groups, scintillation counting orautoradiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent groups and fluorescentgroups. Biotin may be detected using avidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.Standards and standard additions may be used to determine the level ofcadherin in a sample, using well known techniques.

The present invention also provides kits for use in such immunoassays.Such kits generally comprise one or more antibodies, as described above.In addition, one or more additional compartments or containers of a kitgenerally enclose elements, such as reagents, buffers and/or washsolutions, to be used in the immunoassay.

Within further aspects, modulating agents or antibodies (or fragmentsthereof) may be used to facilitate cell identification and sorting invitro or imaging in vivo, permitting the selection of cells expressingdifferent cadherins (or different cadherin levels). Preferably, themodulating agent(s) or antibodies for use in such methods are linked toa detectable marker. Suitable markers are well known in the art andinclude radionuclides, luminescent groups, fluorescent groups, enzymes,dyes, constant immunoglobulin domains and biotin. Within one preferredembodiment, a modulating agent linked to a fluorescent marker, such asfluorescein, is contacted with the cells, which are then analyzed byfluorescence activated cell sorting (FACS).

Antibodies or fragments thereof may also be used within screens ofcombinatorial or other nonpeptide-based libraries to identify othercompounds capable of modulating cadherin-mediated cell adhesion. Suchscreens may generally be performed using an ELISA or other method wellknown to those of ordinary skill in the art that detect compounds with ashape and structure similar to that of the modulating agent. In general,such screens may involve contacting an expression library producing testcompounds with an antibody, and detecting the level of antibody bound tothe candidate compounds. Compounds for which the antibody has a higheraffinity may be further characterized as described herein, to evaluatethe ability to modulate cadherin-mediated cell adhesion.

Identification of HAV-BM Binding Compounds

The present invention further provides methods for identifying compoundsthat bind to an HAV-BM sequence. Such agents may generally be identifiedby contacting a polypeptide as provided herein with a candidate compoundor agent under conditions and for a time sufficient to allow interactionwith a polypeptide comprising an HAV-BM sequence. Any of a variety ofwell known binding assays may then be performed to assess the ability ofthe candidate compound to bind to the polypeptide. In general, acandidate compound that binds to the polypeptide at a significantlygreater level than a similar polypeptide that does not contain an HAV-BMsequence, is considered a compound that binds to an HAV-BM sequence.Preferably, the candidate compound generates a signal within a bindingassay that is at least three standard deviations above the level ofsignal detected for a polypeptide that does not contain an HAV-BMsequence. Depending on the design of the assay, a polypeptide comprisingan HAV-BM sequence may be free in solution, affixed to a solid support,present on a cell surface or located within the cell. Large scalescreens may be performed using automation.

Within certain embodiments, the polypeptide may be immobilized onto asolid support material, and used to affinity purify binding compoundsfrom, for example, cell or tissue extracts. The solid support materialmay be any material known to those of ordinary skill in the art to whichthe polypeptide may be attached. For example, the solid support may be atest well in a microtiter plate or a nitrocellulose filter or othersuitable membrane. Alternatively, the support may be a bead or disc,such as glass, fiberglass, latex or a plastic material such aspolystyrene or polyvinylchloride. The polypeptide may be immobilized onthe solid support using a variety of techniques known to those in theart, which are amply described in the patent and scientific literature.In the context of the present invention, the term “immobilization”refers to both noncovalent association, such as adsorption, and covalentattachment (which may be a direct linkage between the polypeptide andfunctional groups on the support or may be a linkage by way of across-linking agent). Adsorption may be achieved by contacting thepolypeptide, in a suitable buffer, with the solid support for a suitableamount of time. The contact time varies with temperature, but istypically between about 1 hour and 1 day. Covalent attachment ofpolypeptide to a solid support may also generally be achieved by firstreacting the support with a bifunctional reagent that will react withboth the support and a functional group, such as a hydroxyl or aminogroup, on the polypeptide using well known techniques.

Alternatively, a polypeptide may be incubated with whole cells, andinteracting proteins may then be cross-linked to the polypeptide usingstandard techniques. Such polypeptides may be labeled with a detectablemarker (e.g., a radionuclide) or may be subsequently detected using adetection reagent (e.g., an antibody) that is linked to such a marker.Within other assays, cDNA expression libraries may be screened with alabeled polypeptide to identify polynucleotides encoding proteins thatinteract with the labeled polypeptide. Similarly, a yeast two-hybridsystem may be employed to identify interacting proteins. Other assaysmay be performed in a Western blot format, wherein a protein preparationfrom a biological sample such as a cell or tissue extract is submittedto gel electrophoresis, transferred to a suitable membrane and allowedto react with the polypeptide. The presence of the polypeptide on themembrane may then be detected using a label linked to the polypeptide orto a suitable detection reagent, such as an antibody. All of the aboveassays are well known to those of ordinary skill in the art, and may beperformed according to standard protocols. These assays arerepresentative only, and it will be apparent that other assays designedto evaluate binding may also be employed.

Following identification of a compound that binds to an HAV-BM sequence(or a polynucleotide encoding such a compound), standard structuralanalyses may be performed. In general, a polynucleotide may be sequencedusing well known techniques employing such enzymes as Klenow fragment ofDNA polymerase I, Sequenase® (US Biochemical Corp., Cleveland Ohio) Taqpolymerase (Perkin Elmer, Foster City Calif.) or thermostable T7polymerase (Amersham, Chicago, Ill.). An automated sequencing system maybe used, using instruments available from commercial suppliers such asPerkin Elmer and Pharmacia. Proteins may be partially sequenced usingstandard techniques, and the sequence information used to retrieve acDNA molecule encoding the protein (e.g., using PCR or hybridizationscreens employing degenerate oligonucleotides).

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Preparation of Representative Modulating Agents

This Example illustrates the solid phase synthesis of representativepeptide modulating agents.

The peptides were synthesized on a 431A Applied Biosystems peptidesynthesizer using p-Hydroxymethylphenoxymethyl polystyrene (HMP) resinand standard Fmoc chemistry. After synthesis and deprotection, thepeptides were de-salted on a Sephadex G-10 column and lyophilized. Thepeptides were analyzed for purity by analytical HPLC, and in each case asingle peak was observed. Peptides were made as stock solutions at 10 to25 mg/mL in dimethylsulfoxide (DMSO) or water and stored at −20° C.before use.

Example 2 Disruption of the Ability of Mouse Cerebellar Neurons toExtend Neurites

N-cadherin and N-CAM are established as CAMs that can regulate neuriteoutgrowth (Doherty and Walsh, Curr. Op. Neurobiol. 4:49-55, 1994;Williams et al., Neuron 13:583-594, 1994; Hall et al., Cell Adhesion andCommun. 3:441-450, 1996; Doherty and Walsh, Mol. Cell. Neurosci.8:99-111, 1996; Saffell et al., Neuron 18:231-242, 1997). Neuronscultured on monolayers of 3T3 cells that have been transfected withcDNAs encoding N-cadherin or N-CAM extend longer neurites than neuronscultured on the untransfected parental 3T3 cells (commonly referred toas the control 3T3 cells). It has been determined that the neuriteresponse stimulated by transfected N-CAM and N-cadherin initiallydepends upon a trans homophilic binding interaction between thetransfected CAM in the 3T3 cell and the corresponding CAM in the neuron.This Example illustrates the use of representative modulating agents todisrupt neurite outgrowth stimulated by N-cadherin.

Neurons were cultured on monolayers of 3T3 cells transfected with cDNAencoding N-cadherin essentially as described by Doherty and Walsh, Curr.Op. Neurobiol. 4:49-55, 1994; Williams et al., Neuron 13:583-594, 1994;Hall et al., Cell Adhesion and Commun. 3:441-450, 1996; Doherty andWalsh, Mol. Cell. Neurosci. 8:99-111, 1994; Safell et al., Neuron18:231-242, 1997. Briefly, monolayers of control 3T3 fibroblasts and 3T3fibroblasts that express N-cadherin were established by overnightculture of 80,000 cells in individual wells of an 8-chamber well tissueculture slide. 3000 cerebellar neurons isolated from post-natal day 3mouse brains were cultured for 18 hours on the various monolayers incontrol media (SATO/2% FCS), or media supplemented with variousconcentrations of the test peptide to be evaluated. The cultures werethen fixed and stained for GAP43 which specifically binds to the neuronsand their neurites. The length of the longest neurite on each GAP43positive neuron was then measured by computer assisted morphometry. Foreach data point, measurements were made from 100-160 neurons, and thegiven values show the mean +/− the standard error of the mean.

One modulating agent was H-WLKIDPVNGQI-OH (SEQ ID NO:13), an 11 aminoacid peptide containing the ECD4 HAV-BM from human N-cadherin plus someflanking sequence. This peptide is designated N-CAD-CHD2. FIG. 5 showsthe neurite outgrowth response for neurons cultured on monolayers of 3T3cells or 3T3 cells expressing N-cadherin in media containing varyingconcentrations of the modulating agent. In the absence of the peptide,neurites were considerably longer on the N-cadherin monolayers. Thepeptide fully inhibited the N-cadherin response at a concentration of125 and 250 μg/ml.

The mean neurite length was further measured for neurons cultured onmonolayers of 3T3 cells, 3T3 cells expressing N-cadherin, 3T3 cellsexpressing NCAM, and 3T3 cells expressing L1 in media containing thelinear peptide H-WLKIDPVNGQI-OH (SEQ ID NO:13; designated N-CAD-CHD2) ata concentration of 250 μg/ml. The graph shown in FIG. 6 summarizes theresults. Neurite outgrowth was approximately twice as long on 3T3 cellsexpressing either of the cell adhesion molecules N-cadherin, N-CAM, orL1, as compared to outgrowth on 3T3 cells in the absence of the peptide.Only neurite outgrowth on 3T3 cells expressing N-cadherin was inhibitedby the peptide H-WLKIDPVNGQI-OH (SEQ ID NO:13). This peptide istherefore a specific inhibitor of N-cadherin function.

The ability of different modulating agents to inhibit neurite outgrowthwas also evaluated. Table II shows the effects of N-Ac-INPISGQ-NH₂ (SEQID NO:22; derived from EC1 of human N-cadherin), and certain controlpeptides with changes in specific amino acids (N-Ac-INPASGQ-NH₂ (SEQ IDNO:88), N-Ac-INAISGQ-NH₂ (SEQ ID NO:89) and N-Ac-LNPISGQ-NH₂ (SEQ IDNO:90)) on neurite outgrowth. The peptides were tested at differentconcentrations. Rat neurons were grown for 20 hours on monolayers ofeither 3T3 cells, or 3T3 cells expressing N-cadherin. The cultures werethen fixed, and the mean neurite length was determined by makingmeasurements on at least 150 neurons for each treatment. The results arepresented as the percentage inhibition of neurite outgrowth over 3T3cells expressing N-cadherin. The peptides did not inhibit neuriteoutgrowth over 3T3 cells not expressing N-cadherin. TABLE II Effect(Percent Inhibition) of Representative Modulating Agents on NeuriteOutgrowth on 3T3 Cells Expressing N-cadherin Sequence 100 μg/ml 33 μg/ml10 μg/ml 3 μg/ml N-Ac-INPISGQ-NH₂ 94.5 ± 4.8 73.0 ± 4.6 47.8 ± 4.1 19.5± 4.3 (SEQ ID NO: 22) N-Ac-INPASGQ-NH₂  7.2 ± 3.2 (SEQ ID NO: 88)N-Ac-INAISGQ-NH₂ 37.2 ± 2.3 12.3 ± 6.2 (SEQ ID NO: 89) N-Ac-LNPISGQ-NH₂19.2 ± 7.7 (SEQ ID NO: 90)

Similar experiments, illustrated in Table III, were performed toevaluate the effect of various cyclic peptide modulating agents derivedfrom EC1 of human N-cadherin on neurite outgrowth. TABLE III Effect ofCyclic Peptide Modulating Agents on Neurite Outgrowth on 3T3 CellsExpressing N-cadherin EC₅₀ Percent Inhibition of Neurite OutgrowthSequence (mM) 100 μg/ml 33 μg/ml 10 μg/ml 3 μg/ml N-Ac-CINPC-NH₂ 0.014398.1 ± 8.2 71.0 ± 6.5 51.9 ± 2.5 17.4 ± 5.7 (SEQ ID NO: 86)N-Ac-CINPIC-NH₂ 0.0382  94.5 ± 10.5 56.2 ± 6.9 31.5 ± 7.5  7.5 ± 3.2(SEQ ID NO: 87) N-Ac-CINPISC-NH₂ 0.0381 73.4 ± 2.2 39.5 ± 4.3 21.8 ± 1.2(SEQ ID NO: 63)

To further evaluate the specificity of these peptide modulating agents,inhibition of neurite outgrowth was assessed, at 100 μg/ml, onmonolayers of 3T3 cells transfected with various molecules responsiblefor neurite outgrowth. The results, shown in Table IV, illustrate a highlevel of specificity for N-cadherin mediated neurite outgrowth. TABLE IVEffect (Percent Inhibition) of Representative Peptide Modulating Agentson Neurite Outgrowth over 3T3 Cells Expressing Various Protein Involvedin Neurite Outgrowth N-Ac-INPISGQ-NH₂ N-Ac-CINPIC-NH₂ TransfectedProtein (SEQ ID NO: 22) (SEQ ID NO: 86) N-cadherin 94.5 ± 4.8  94.5 ±10.5 NCAM 6.2 ± 3.0 13.7 ± 7.1  FGFR 1.9 ± 5.1 L1 0.3 ± 1.8 7.8 ± 3.8

Further experiments were performed as described above to assess theactivity of peptide modulating agents derived from EC4 of humanN-cadherin. Table V shows the effect on neurite outgrowth of agentscomprising the sequence IDPVN (SEQ ID NO:29), as well as agentscontaining substitutions within this sequence. The EC₅₀ (mM) forN-Ac-WLKIDPVNGQI-NH₂ (SEQ ID NO:13) was 0.046. TABLE V Effect of PeptideModulating Agents on Neurite Outgrowth Percent Inhibition of NeuriteOutgrowth Sequence 250 μg/ml 100 μg/ml 33 μg/ml 11 μg/mlN-Ac-WLKIDPVNGQI-NH₂ 83.5 ± 6.6 66.9 ± 3.7 19.8 ± 5.0 (SEQ ID NO: 13)N-Ac-WLKADPVNGQI-NH₂ 54.7 ± 1.2  22.2 ± 11.0 (SEQ ID NO: 91)N-Ac-WLKIDAVNGQI-NH₂  52.3 ± 10.3 12.4 ± 7.6 (SEQ ID NO: 92)N-Ac-WLKADAVNGQI-NH₂ 19.7 ± 5.2 10.5 ± 4.1 (SEQ ID NO: 93)N-Ac-IDPVNGQ-NH₂ 85.6 ± 7.3 56.4 ± 7.7 34.3 ± 2.4  0.6 ± 10.0 (SEQ IDNO: 94) H-WLKIDPVNGQI-OH 76.1 ± 7.3 (SEQ ID NO: 13) N-Ac-NLKIDPVNGQI-NH₂86.8 ± 8.2 67.8 ± 7.7 25.6 ± 6.7 H-LKIDPVNGQI-OH  46.0 ± 10.0 (SEQ IDNO: 21) H-LKIDPANGQI-OH 56.8 ± 1.2 (SEQ ID NO: 64) H-LKIDAVNGQI-OH 103.8± 8.8  (SEQ ID NO: 65) N-Ac-CIDPVNC-NH₂ 96.4 ± 7.3 75.8 ± 2.0 40.6 ± 6.315.4 ± 8.9 (SEQ ID NO: 62)

These results demonstrate that modulating agents comprising an HAV-BMsequence are effective and specific inhibitors of N-cadherin function.

Example 3 Modulating Agent Binding to N-Cadherin

This Example illustrates the ability of a representative modulatingagent to bind to N-cadherin.

The peptide H-WLKIDPVNGQI-OH (SEQ ID NO:13) was passed over flow cellscoated with an N-cadherin-Fc chimera or human IgG1 at a concentration ofeither 250, 500 or 1000 μg/ml. FIG. 7 is a graph illustrating theassociation of the peptide to the flow cell coated with the N-cadherinFc chimera, with the binding to the control flow cell (coated with humanIgG1) automatically subtracted.

Example 4 Effect of a Representative Modulating Agent on Tumor CellAdhesion

This Example illustrates the ability of a modulating agent to disrupttumor cell adhesion.

Monolayer cultures of human ovarian cancer cells (SKOV3) were grown inthe presence and absence of the peptide N-Ac-INPISGQ-NH₂ (SEQ ID NO:22).FIG. 8A shows the cells grown in the absence of peptide. FIG. 8B showsthe cells 24 hours after being cultured in the presence of 1 mg/mL ofN-Ac-INPISGQ (SEQ ID NO:22). The SKOV3 cells retract from one anotherand round-up when cultured in the presence of the peptide.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1.-62. (canceled)
 63. A method for modulating a cadherin-mediatedfunction, comprising contacting a cadherin-expressing cell with a celladhesion modulating agent comprising at least three amino acid residuesof the HAV-BM sequence VFSINSMSGRM (SEQ ID NO:8), wherein the agent iseffective for inhibiting a cadherin-mediated function.
 64. A method formodulating a cadherin-mediated function, comprising contacting acadherin-expressing cell with a cell adhesion modulating agentcomprising at least three amino acid residues of the HAV-BM sequenceVFSINSMSGRM (SEQ ID NO:8), wherein the agent is a peptide ranging insize from three to seven amino acid residues, and wherein the agent iseffective for inhibiting a cadherin-mediated function.
 65. A method formodulating a cadherin-mediated function, comprising contacting acadherin-expressing cell with a cell adhesion modulating agentcomprising at least three amino acid residues of the HAV-BM sequenceVFSINSMSGRM (SEQ ID NO:8), wherein the agent is a peptide ranging insize from six to sixteen amino acid residues, and wherein the agent iseffective for inhibiting a cadherin-mediated function.
 66. A method formodulating a cadherin-mediated function, comprising contacting acadherin-expressing cell with a cell adhesion modulating agentcomprising an antibody or antigen binding fragment thereof that binds tothe HAV-BM sequence VFSINSMSGRM (SEQ ID NO:8), wherein the agent iseffective for inhibiting a cadherin-mediated function.
 67. A methodaccording to any one of claims 63-65, wherein the agent comprises anHAV-BM sequence that is present within a linear peptide.
 68. A methodaccording to any one of claims 63-65, wherein the agent comprises anHAV-BM sequence that is present within a cyclic peptide.
 69. A methodaccording to any one of claims 63-65, wherein the HAV-BM sequence ispresent within a peptide comprising an N-terminal or C-terminalmodification.